Cytosolic Isobutanol Pathway Localization for the Production of Isobutanol

ABSTRACT

The present invention provides recombinant microorganisms comprising isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol, wherein said recombinant microorganism is selected to produce isobutanol from a carbon source. Methods of using said recombinant microorganisms to produce isobutanol are also provided. In various aspects of the invention, the recombinant microorganisms may comprise a cytosolically active isobutanol pathway enzymes. In some embodiments, the invention provides mutated, modified, and/or chimeric isobutanol pathway enzymes with cytosolic activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the  Saccharomyces  clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/176,452, filed Jul. 5, 2011, which is a divisional of U.S.application Ser. No. 12/855,276, filed Aug. 12, 2010, which issued asU.S. Pat. No. 8,232,089, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/272,058, filed Aug. 12, 2009, and U.S.Provisional Application Ser. No. 61/272,059, filed Aug. 12, 2009, eachof which are herein incorporated by reference in their entireties forall purposes.

ACKNOWLEDGMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Contract No.IIP-0823122, awarded by the National Science Foundation, and underContract No. EP-D-09-023, awarded by the Environmental ProtectionAgency. The government has certain rights in the invention.

TECHNICAL FIELD

Recombinant microorganisms and methods of producing such organisms areprovided. Also provided are methods of producing metabolites that arebiofuels by contacting a suitable substrate with recombinantmicroorganisms and enzymatic preparations therefrom.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:GEVO_(—)041_(—)18US_SeqList_ST25.txt, date recorded: Jan. 16, 2014, filesize: 343 kilobytes).

BACKGROUND

Biofuels have a long history ranging back to the beginning of the 20thcentury. As early as 1900, Rudolf Diesel demonstrated at the WorldExhibition in Paris, France, an engine running on peanut oil. Soonthereafter, Henry Ford demonstrated his Model T running on ethanolderived from corn. Petroleum-derived fuels displaced biofuels in the1930s and 1940s due to increased supply, and efficiency at a lower cost.

Market fluctuations in the 1970s coupled to the decrease in US oilproduction led to an increase in crude oil prices and a renewed interestin biofuels. Today, many interest groups, including policy makers,industry planners, aware citizens, and the financial community, areinterested in substituting petroleum-derived fuels with biomass-derivedbiofuels. The leading motivations for developing biofuels are ofeconomical, political, and environmental nature.

One is the threat of ‘peak oil’, the point at which the consumption rateof crude oil exceeds the supply rate, thus leading to significantlyincreased fuel cost results in an increased demand for alternativefuels. In addition, instability in the Middle East and other oil-richregions has increased the demand for domestically produced biofuels.Also, environmental concerns relating to the possibility of carbondioxide related climate change is an important social and ethicaldriving force which is starting to result in government regulations andpolicies such as caps on carbon dioxide emissions from automobiles,taxes on carbon dioxide emissions, and tax incentives for the use ofbiofuels.

Ethanol is the most abundant fermentatively produced fuel today but hasseveral drawbacks when compared to gasoline. Butanol, in comparison, hasseveral advantages over ethanol as a fuel: it can be made from the samefeedstocks as ethanol but, unlike ethanol, it is compatible withgasoline at any ratio and can also be used as a pure fuel in existingcombustion engines without modifications. Unlike ethanol, butanol doesnot absorb water and can thus be stored and distributed in the existingpetrochemical infrastructure. Due to its higher energy content which isclose to that of gasoline, the fuel economy (miles per gallon) is betterthan that of ethanol. Also, butanol-gasoline blends have lower vaporpressure than ethanol-gasoline blends, which is important in reducingevaporative hydrocarbon emissions.

Isobutanol has the same advantages as butanol with the additionaladvantage of having a higher octane number due to its branched carbonchain. Isobutanol is also useful as a commodity chemical and is also aprecursor to MTBE.

Isobutanol has been produced in recombinant microorganisms expressing aheterologous, five-step metabolic pathway (See, e.g., WO/2007/050671 toDonaldson et al., WO/2008/098227 to Liao et al., and WO/2009/103533 toFestel et al.). However, the microorganisms produced have fallen shortof commercial relevance due to their low performance characteristics,including, for example low productivity, low titer, low yield, and therequirement for oxygen during the fermentation process. Thus,recombinant microorganisms exhibiting increased isobutanol productivity,titer, and/or yield are desirable.

SUMMARY OF THE INVENTION

The present invention provides cytosolically active dihydroxyaciddehydratase (DHAD) enzymes and recombinant microorganisms comprisingsaid cytosolically active DHAD enzymes. In some embodiments, saidrecombinant microorganisms may further comprise one or more additionalenzymes catalyzing a reaction in an isobutanol producing metabolicpathway. As described herein, the recombinant microorganisms of thepresent invention are useful for the production of several beneficialmetabolites, including, but not limited to isobutanol.

In a first aspect, the invention provides cytosolically activedihydroxyacid dehydratase (DHAD) enzymes. These cytosolically activeDHAD enzymes generally exhibit the ability to convert2,3-dihydroxyisovalerate to ketoisovalerate in the cytosol. Thecytosolically active DHAD enzymes of the present invention, as describedherein, can include native (i.e. parental) DHAD enzymes that exhibitcytosolic activity, as well DHAD enzymes that have been modified ormutated to increase their cytosolic localization and/or activity ascompared to native (i.e. parental) DHAD enzymes.

In various embodiments described herein, the DHAD enzymes may be derivedfrom a prokaryotic organism. In one embodiment, the prokaryotic organismis a bacterial organism. In another embodiment, the bacterial organismis Lactococcus lactis. In a specific embodiment, the DHAD enzyme from L.lactis comprises the amino acid sequence of SEQ ID NO: 18. In anotherembodiment, the bacterial organism is Francisella tularensis. In aspecific embodiment, the DHAD enzyme from F. tularensis comprises theamino acid sequence of SEQ ID NO: 14. In another embodiment, thebacterial organism is Gramella forsetii. In a specific embodiment, theDHAD enzyme from G. forsetii comprises the amino acid sequence of SEQ IDNO: 17.

In alternative embodiments described herein, the DHAD enzyme may bederived from a eukaryotic organism. In one embodiment, the eukaryoticorganism is a fungal organism. In an exemplary embodiment, the fungalorganism is Neurospora crassa. In a specific embodiment, the DHAD enzymefrom N. crassa comprises the amino acid sequence of SEQ ID NO: 165.

In some embodiments, the invention provides modified or mutated DHADenzymes, wherein said DHAD enzymes exhibit increased cytosolic activityas compared to their parental DHAD enzymes. In another embodiment, theinvention provides modified or mutated DHAD enzymes, wherein said DHADenzymes exhibit increased cytosolic activity as compared to the DHADenzyme comprised by the amino acid sequence of SEQ ID NO: 11.

In further embodiments, the invention provides DHAD enzymes comprisingthe amino acid sequence P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27), wherein Xis any natural or non-natural amino acid, and wherein said DHAD enzymesexhibit the ability to convert 2,3-dihydroxyisovalerate toketoisovalerate in the cytosol.

In some embodiments, the DHAD enzymes of the present invention exhibit aproperly folded iron-sulfur cluster domain and/or redox active domain inthe cytosol. In one embodiment, the DHAD enzymes comprise a mutated ormodified iron-sulfur cluster domain and/or redox active domain.

In another aspect, the present invention provides recombinantmicroorganisms comprising a cytosolically active DHAD enzyme. In oneembodiment, the invention provides recombinant microorganisms comprisinga DHAD enzyme derived from a prokaryotic organism, wherein said DHADenzyme exhibits activity in the cytosol. In one embodiment, the DHADenzyme is derived from a bacterial organism. In a specific embodiment,the DHAD enzyme is derived from L. lactis and comprises the amino acidsequence of SEQ ID NO: 18. In another embodiment, the invention providesrecombinant microorganisms comprising a DHAD enzyme derived from aeukaryotic organism, wherein said DHAD enzyme exhibits activity in thecytosol. In one embodiment, the DHAD enzyme is derived from a fungalorganism. In an alternative embodiment, the DHAD enzyme is derived froma yeast organism.

In one embodiment, the invention provides recombinant microorganismscomprising a modified or mutated DHAD enzyme, wherein said DHAD enzymeexhibits increased cytosolic activity as compared to the parental DHADenzyme. In another embodiment, the invention provides recombinantmicroorganisms comprising a modified or mutated DHAD enzyme, whereinsaid DHAD enzyme exhibits increased cytosolic activity as compared tothe DHAD enzyme comprised by the amino acid sequence of SEQ ID NO: 11.

In another embodiment, the invention provides recombinant microorganismscomprising a DHAD enzyme comprising the amino acid sequenceP(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27), wherein X is any natural ornon-natural amino acid, and wherein said DHAD enzymes exhibit theability to convert 2,3-dihydroxyisovalerate to ketoisovalerate in thecytosol.

In some embodiments, the invention provides recombinant microorganismscomprising a DHAD enzyme fused to a peptide tag, whereby said DHADenzyme exhibits increased cytosolic localization and/or cytosolic DHADactivity as compared to the parental microorganism. In one embodiment,the peptide tag is non-cleavable. In another embodiment, the peptide tagis fused at the N-terminus of the DHAD enzyme. In another embodiment,the peptide tag is fused at the C-terminus of the DHAD enzyme. Incertain embodiments, the peptide tag may be selected from the groupconsisting of ubiquitin, ubiquitin-like (UBL) proteins, myc, HA-tag,green fluorescent protein (GFP), and the maltose binding protein (MBP).

In certain embodiments described herein, it may be desirable to furtheroverexpress an additional enzyme that converts 2,3-dihydroxyisovalerate(DHIV) to ketoisovalerate (KIV) in the cytosol. In a specificembodiment, the enzyme may be selected from the group consisting of3-isopropylmalate isomerase (Leu1p) and imidazoleglycerol-phosphatedehydrogenase (His3p).

In various embodiments described herein, the recombinant microorganismsmay be further engineered to express an isobutanol producing metabolicpathway comprising at least one exogenous gene that catalyzes a step inthe conversion of pyruvate to isobutanol. In one embodiment, therecombinant microorganism may be engineered to express an isobutanolproducing metabolic pathway comprising at least two exogenous genes. Inanother embodiment, the recombinant microorganism may be engineered toexpress an isobutanol producing metabolic pathway comprising at leastthree exogenous genes. In another embodiment, the recombinantmicroorganism may be engineered to express an isobutanol producingmetabolic pathway comprising at least four exogenous genes. In anotherembodiment, the recombinant microorganism may be engineered to expressan isobutanol producing metabolic pathway comprising five exogenousgenes. Thus, the present invention further provides recombinantmicroorganisms that comprise an isobutanol producing metabolic pathwayand methods of using said recombinant microorganisms to produceisobutanol.

In one embodiment, the recombinant microorganisms comprise an isobutanolproducing metabolic pathway with at least one isobutanol pathway enzymelocalized in the cytosol. In another embodiment, the recombinantmicroorganisms comprise an isobutanol producing metabolic pathway withat least two isobutanol pathway enzymes localized in the cytosol. Inanother embodiment, the recombinant microorganisms comprise anisobutanol producing metabolic pathway with at least three isobutanolpathway enzymes localized in the cytosol. In another embodiment, therecombinant microorganisms comprise an isobutanol producing metabolicpathway with at least four isobutanol pathway enzymes localized in thecytosol. In an exemplary embodiment, the recombinant microorganismscomprise an isobutanol producing metabolic pathway with five isobutanolpathway enzymes localized in the cytosol. In a further exemplaryembodiment, at least one of the pathway enzymes localized to the cytosolis a cytosolically active DHAD enzyme as disclosed herein.

In various embodiments described herein, the isobutanol pathwayenzyme(s) is/are selected from the group consisting of acetolactatesynthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxyaciddehydratase (DHAD), 2-keto-acid decarboxylase (KIVD), and alcoholdehydrogenase (ADH).

As described herein, the cytosolically active isobutanol pathway enzymesof the present invention can include native (i.e. parental) enzymes thatexhibit cytosolic activity, as well isobutanol pathway enzymes that havebeen modified or mutated to increase their cytosolic localization and/oractivity as compared to native (i.e. parental) pathway enzymes.

In various embodiments described herein, the isobutanol pathway enzymesmay be derived from a prokaryotic organism. In alternative embodimentsdescribed herein, the isobutanol pathway enzymes may be derived from aeukaryotic organism.

In some embodiments, the invention provides modified or mutatedisobutanol pathway enzymes, wherein said isobutanol pathway enzymesexhibit increased cytosolic activity as compared to their parentalisobutanol pathway enzymes. In another embodiment, the inventionprovides modified or mutated isobutanol pathway enzymes, wherein saidisobutanol pathway enzymes exhibit increased cytosolic activity ascompared to the homologous isobutanol pathway enzyme from S. cerevisiae.

In various embodiments described herein, at least one of the isobutanolpathway enzymes exhibiting cytosolic activity is ALS. In one embodiment,the ALS is derived from a prokaryotic organism, including, but notlimited to Bacillus subtilis or L. lactis. In another embodiment, theALS is derived from a eukaryotic organism, including, but not limited toMagnaporthe grisea, Phaeosphaeria nodorum, Talaromyces stipitatus, andTrichoderma atroviride.

In additional embodiments, at least one of the isobutanol pathwayenzymes exhibiting cytosolic activity is KARI. In one embodiment, theKARI is derived from a prokaryotic organism, including, but not limitedto Escherichia coli, B. subtilis or L. lactis. In another embodiment,the KARI is derived from a eukaryotic organism, including, but notlimited to Piromyces sp. E2, S. cerevisiae, and Arabidopsis. In certainspecific embodiments, the KARI comprises an amino acid sequence selectedfrom an organism selected from the group consisting of E. coli, S.cerevisiae, B. subtilis Piromyces sp. E2, Buchnera aphidicola, Spinaciaoleracea, Oryza sativa, Chlamydomonas reinhardtii, N. crassa,Schizosaccharomyces pombe, Laccaria bicolor, Ignicoccus hospitalis,Picrophilus torridus, Acidiphilium cryptum, Cyanobacteria/Synechococcussp., Zymomonas mobilis, Bacteroides thetaiotaomicron, Methanococcusmaripaludis, Vibrio fischeri, Shewanella sp, G. forsetii, Psychromonasingrhamaii, and Cytophaga hutchinsonii. In additional embodiments, theKARI may be an NADH-dependent KARI.

In various embodiments described herein, the isobutanol pathway enzymemay be mutated or modified to remove an N-terminal mitochondrialtargeting sequence (MTS). Removal of the MTS can increase cytosoliclocalization of the isobutanol pathway enzyme and/or increase thecytosolic activity of the isobutanol pathway enzyme as compared to theparental isobutanol pathway enzyme.

In some embodiments, the MTS may be modified or mutated to reduce oreliminate its ability to target the isobutanol pathway enzyme to themitochondria. Selected modification of the MTS can increase cytosoliclocalization of the isobutanol pathway enzyme and/or increase thecytosolic activity of the isobutanol pathway enzyme as compared to theparental isobutanol pathway enzyme.

In additional embodiments, the invention provides recombinantmicroorganisms comprising an isobutanol pathway enzyme fused to apeptide tag, whereby said isobutanol pathway enzyme exhibits increasedcytosolic localization and/or cytosolic activity as compared to theparental enzyme. As a result, the recombinant microorganism comprisingthe tagged isobutanol pathway enzyme will generally exhibit an increasedability to perform a step involved in the conversion of pyruvate toisobutanol in the cytosol. In one embodiment, the peptide tag isnon-cleavable. In another embodiment, the peptide tag is fused at theN-terminus of the isobutanol pathway enzyme. In another embodiment, thepeptide tag is fused at the C-terminus of the isobutanol pathway enzyme.In certain embodiments, the peptide tag may be selected from the groupconsisting of ubiquitin, ubiquitin-like (UBL) proteins, myc, HA-tag,green fluorescent protein (GFP), and the maltose binding protein (MBP).

In various embodiments described herein, the recombinant microorganismsmay further comprise a nucleic acid encoding a chaperone protein,wherein said chaperone protein assists the folding of a proteinexhibiting cytosolic activity. In a preferred embodiment, the proteinexhibiting cytosolic activity is an isobutanol pathway enzyme. In oneembodiment, the chaperone may be a native protein. In anotherembodiment, the chaperone protein may be an exogenous protein. In someembodiments, the chaperone protein may be selected from the groupconsisting of: endoplasmic reticulum oxidoreductin 1 (Ero1) includingvariants of Ero1 that have been suitably altered to reduce or preventits normal localization to the endoplasmic reticulum; thioredoxins(including, but not limited to, Trx1 and Trx2), thioredoxin reductase(Trr1), glutaredoxins (including, but not limited to, Grx1, Grx2, Grx3,Grx4, Grx5, Grx6, Grx7, and Grx8), glutathione reductase (Gir1), andJac1, including variants of Jac1 that have been suitably altered toreduce or prevent its normal mitochondrial localization; and homologs orvariants thereof.

In some embodiments, the recombinant microorganisms may further compriseone or more genes encoding an iron-sulfur cluster assembly protein. Inone embodiment, the iron-sulfur cluster assembly protein encoding genesmay be derived from prokaryotic organisms. In one embodiment, theiron-sulfur cluster assembly protein encoding genes are derived from abacterial organism, including, but not limited to E. coli, L. lactis,Helicobacter pylori, and Entamoeba histolytica. In specific embodiments,the bacterially derived iron-sulfur cluster assembly protein encodinggenes are selected from the group consisting of cyaY, iscS, iscU, iscA,hscB, hscA, fdx, isuX, sufA, sufB, sufC, sufD, sufS, sufE, apbC, andhomologs or variants thereof.

In another embodiment, the iron-sulfur cluster assembly protein encodinggenes may be derived from eukaryotic organisms, including, but notlimited to yeasts and plants. In one embodiment, the iron-sulfur clusterprotein encoding genes are derived from a yeast organism, including, butnot limited to S. cerevisiae. In specific embodiments, the yeast derivedgenes encoding iron-sulfur cluster assembly proteins are selected fromthe group consisting of Cfd1, Nbp35, Nar1, Cia1, and homologs orvariants thereof. In a further embodiment, the iron-sulfur clusterassembly protein encoding genes may be derived from plant nuclear geneswhich encode proteins translocated to chloroplast or plant genes foundin the chloroplast genome itself.

In some embodiments, one or more genes encoding an iron-sulfur clusterassembly protein may be mutated or modified to remove a signal peptide,whereby localization of the product of said one or more genes to themitochondria or other subcellular compartment is prevented. In certainembodiments, it may be preferable to overexpress one or more genesencoding an iron-sulfur cluster assembly protein.

In certain embodiments described herein, it may be desirable to reduceor eliminate the activity and/or proteins levels of one or moreiron-sulfur cluster containing cytosolic proteins. In a specificembodiment, the iron-sulfur cluster containing cytosolic protein is3-isopropylmalate dehydratase (Leu1p). In one embodiment, therecombinant microorganism comprises a mutation in the LEU1 generesulting in the reduction of Leu1p protein levels. In anotherembodiment, the recombinant microorganism comprises a partial deletionin the LEU1 gene resulting in the reduction of Leu1p protein levels. Inanother embodiment, the recombinant microorganism comprises a completedeletion in the LEU1 gene resulting in the reduction of Leu1p proteinlevels. In another embodiment, the recombinant microorganism comprises amodification of the regulatory region associated with the LEU1 generesulting in the reduction of Leu1p protein levels. In yet anotherembodiment, the recombinant microorganism comprises a modification of atranscriptional regulator for the LEU1 gene resulting in the reductionof Leu1p protein levels.

In additional embodiments, the present invention provides recombinantmicroorganisms comprising chimeric proteins consisting of isobutanolpathway enzymes. In one embodiment, the chimeric proteins consist of ALSand at least one additional protein. In a specific embodiment, theadditional protein is KARI. In a preferred embodiment, the chimericprotein exhibits the biocatalytic properties of both ALS and KARI. Sucha chimeric protein allows for an increase in the concentration of2-acetolactate at the active site of KARI as compared to the parentalmicroorganism, giving the recombinant microorganism an enhanced abilityto convert 2-acetolactate to 2,3-dihydroxyisovalerate. In anotherembodiment, the chimeric proteins consist of KARI and at least oneadditional protein. In a specific embodiment, the additional protein isDHAD. In a preferred embodiment, the chimeric protein exhibits thebiocatalytic properties of both KARI and DHAD. In each of the variousembodiments described herein, the proteins may be connected via aflexible linker.

In various embodiments described herein, the recombinant microorganismsmay be engineered to express native genes that catalyze a step in theconversion of pyruvate to isobutanol. In one embodiment, the recombinantmicroorganism is engineered to increase the activity of a nativemetabolic pathway gene for conversion of pyruvate to isobutanol. Inanother embodiment, the recombinant microorganism is further engineeredto include at least one enzyme encoded by an exogenous gene and at leastone enzyme encoded by a native gene. In yet another embodiment, therecombinant microorganism comprises a reduction in the activity of anative metabolic pathway as compared to a parental microorganism.

In another embodiment, the present invention provides recombinantmicroorganisms comprising a scaffold system tethered to one or moreisobutanol pathway enzymes. In a specific embodiment, the scaffoldsystem is the MAP kinase scaffold (Ste5) system. In a furtherembodiment, one or more of the isobutanol pathway enzymes may bemodified or mutated to comprise a protein domain allowing for binding tothe scaffold system.

In various embodiments described herein, the recombinant microorganismsmay be microorganisms of the Saccharomyces clade, Saccharomyces sensustricto microorganisms, Crabtree-negative yeast microorganisms,Crabtree-positive yeast microorganisms, post-WGD (whole genomeduplication) yeast microorganisms, pre-WGD (whole genome duplication)yeast microorganisms, and non-fermenting yeast microorganisms.

In some embodiments, the recombinant microorganisms may be yeastrecombinant microorganisms of the Saccharomyces clade.

In some embodiments, the recombinant microorganisms may be Saccharomycessensu stricto microorganisms. In one embodiment, the Saccharomyces sensustricto is selected from the group consisting of S. cerevisiae, S.kudriavzevii, S. mikatae, S. bayanus, S. uvarum. S. carocanis andhybrids thereof.

In some embodiments, the recombinant microorganisms may beCrabtree-negative recombinant yeast microorganisms. In one embodiment,the Crabtree-negative yeast microorganism is classified into a generaselected from the group consisting of Kluyveromyces, Pichia, Hansenula,Issatchenkia, or Candida. In additional embodiments, theCrabtree-negative yeast microorganism is selected from Kluyveromyceslactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis,Hansenula anomala, Issatchenkia orientalis, Candida utilis andKluyveromyces waltii.

In some embodiments, the recombinant microorganisms may beCrabtree-positive recombinant yeast microorganisms. In one embodiment,the Crabtree-positive yeast microorganism is classified into a generaselected from the group consisting of Saccharomyces, Kluyveromyces,Zygosaccharomyces, Debaryomyces, Candida, Pichia andSchizosaccharomyces. In additional embodiments, the Crabtree-positiveyeast microorganism is selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus,Saccharomyces paradoxus, Saccharomyces castelli, Saccharomyces kluyveri,Kluyveromyces thermotolerans, Candida glabrata, Z. bailli, Z. rouxii,Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, andSaccharomyces uvarum.

In some embodiments, the recombinant microorganisms may be post-WGD(whole genome duplication) yeast recombinant microorganisms. In oneembodiment, the post-WGD yeast recombinant microorganism is classifiedinto a genera selected from the group consisting of Saccharomyces orCandida. In additional embodiments, the post-WGD yeast is selected fromthe group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum,Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli,and Candida glabrata.

In some embodiments, the recombinant microorganisms may be pre-WGD(whole genome duplication) yeast recombinant microorganisms. In oneembodiment, the pre-WGD yeast recombinant microorganism is classifiedinto a genera selected from the group consisting of Saccharomyces,Kluyveromyces, Candida, Pichia, Debaryomyces, Hansenula, Pachysolen,Yarrowia, Issatchenkia, and Schizosaccharomyces. In additionalembodiments, the pre-WGD yeast is selected from the group consisting ofSaccharomyces kluyveri, Kluyveromyces thermotolerans, Kluyveromycesmarxianus, Kluyveromyces waltii, Kluyveromyces lactis, Candidatropicalis, Pichia pastoris, Pichia anomala, Pichia stipitis,Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis,Yarrowia lipolytica, Issatchenkia orientalis, and Schizosaccharomycespombe.

In some embodiments, the recombinant microorganisms may bemicroorganisms that are non-fermenting yeast microorganisms, including,but not limited to those, classified into a genera selected from thegroup consisting of Tricosporon, Rhodotorula, or Myxozyma.

In another aspect, the present invention provides methods of producingisobutanol using one or more recombinant microorganisms of theinvention. In one embodiment, the method includes cultivating one ormore recombinant microorganisms in a culture medium containing afeedstock providing the carbon source until a recoverable quantity ofthe isobutanol is produced and optionally, recovering the isobutanol. Inone embodiment, the microorganism is selected to produce isobutanol froma carbon source at a yield of at least about 5 percent theoretical. Inanother embodiment, the microorganism is selected to produce isobutanolat a yield of at least about 10 percent, at least about 15 percent,about least about 20 percent, at least about 25 percent, at least about30 percent, at least about 35 percent, at least about 40 percent, atleast about 45 percent, at least about 50 percent, at least about 55percent, at least about 60 percent, at least about 65 percent, at leastabout 70 percent, at least about 75 percent, at least about 80 percenttheoretical, at least about 85 percent theoretical, or at least about 90percent theoretical.

In one embodiment, the microorganism produces isobutanol from a carbonsource at a specific productivity of at least about 0.7 mg/L/hr per OD.In another embodiment, the microorganism produces isobutanol from acarbon source at a specific productivity of at least about 1 mg/L/hr perOD, at least about 10 mg/L/hr per OD, at least about 50 mg/L/hr per OD,at least about 100 mg/L/hr per OD, at least about 250 mg/L/hr per OD, orat least about 500 g/L/hr per OD.

BRIEF DESCRIPTION OF DRAWINGS

Illustrative embodiments of the invention are illustrated in thedrawings, in which:

FIG. 1 illustrates an exemplary embodiment of an isobutanol pathway.

FIG. 2 illustrates acetoin produced from GEVO 1187 (no ALS), 2280 (B.subtilis AlsS not codon optimized), GEVO 2618 (B. subtilis AlsS), GEVO2621 (T. atroviride ALS) and GEVO 2622 (T. stipitatus ALS). All acetoinvalues are normalized to OD₆₀₀ and reported as mM/OD.

FIG. 3 illustrates the specific activity at pH 7.5 of KARI enzyme inwhole cell lysates for GEVO1803 containing empty vector (pGV1102),ilv5ΔN47(pGV1831), ilv5ΔN46(pGV1901), Full length ILV5 (pGV1833) and E.coli ilvC codon optimized for S. cerevisiae (pGV1824).

FIG. 4 illustrates the results from fermentations of GEVO2107transformed with plasmids for expression of KARI and different DHADhomologs (shown in legend).

FIG. 5 illustrates a phylogenetic tree of 53 representative DHADhomologs following pairwise global alignments and progressive assemblyof alignments using Neighbor-Joining phylogeny.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a polynucleotide” includes aplurality of such polynucleotides and reference to “the microorganism”includes reference to one or more microorganisms, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

Any publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The term “microorganism” includes prokaryotic and eukaryotic microbialspecies from the Domains Archaea, Bacteria and Eucarya, the latterincluding yeast and filamentous fungi, protozoa, algae, or higherProtista. The terms “microbial cells” and “microbes” are usedinterchangeably with the term microorganism.

The term “genus” is defined as a taxonomic group of related speciesaccording to the Taxonomic Outline of Bacteria and Archaea (Garrity etal., 2007, TOBA Release 7.7, Michigan State University Board ofTrustees).

The term “species” is defined as a collection of closely relatedorganisms with greater than 97% 16S ribosomal RNA sequence homology andgreater than 70% genomic hybridization and sufficiently different fromall other organisms so as to be recognized as a distinct unit.

The terms “recombinant microorganism,” “modified microorganism” and“recombinant host cell” are used interchangeably herein and refer tomicroorganisms that have been genetically modified to express orover-express endogenous polynucleotides, or to express heterologouspolynucleotides, such as those included in a vector, or which have analteration in expression of an endogenous gene. By “alteration” it ismeant that the expression of the gene, or level of a RNA molecule orequivalent RNA molecules encoding one or more polypeptides orpolypeptide subunits, or activity of one or more polypeptides orpolypeptide subunits is up regulated or down regulated, such thatexpression, level, or activity is greater than or less than thatobserved in the absence of the alteration. For example, the term “alter”can mean “inhibit,” but the use of the word “alter” is not limited tothis definition.

The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein results from transcription andtranslation of the open reading frame sequence. The level of expressionof a desired product in a host cell may be determined on the basis ofeither the amount of corresponding mRNA that is present in the cell, orthe amount of the desired product encoded by the selected sequence. Forexample, mRNA transcribed from a selected sequence can be quantitated byqRT-PCR or by Northern hybridization (Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press(1989)). Protein encoded by a selected sequence can be quantitated byvarious methods, e.g., by ELISA, by assaying for the biological activityof the protein, or by employing assays that are independent of suchactivity, such as western blotting or radioimmunoassay, using antibodiesthat recognize and bind the protein. The polynucleotide generallyencodes a target enzyme involved in a metabolic pathway for producing adesired metabolite. It is understood that the terms “recombinantmicroorganism” and “recombinant host cell” refer not only to theparticular recombinant microorganism but to the progeny or potentialprogeny of such a microorganism. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

The term “wild-type microorganism” describes a cell that occurs innature, i.e. a cell that has not been genetically modified. A wild-typemicroorganism can be genetically modified to express or overexpress afirst target enzyme. This microorganism can act as a parentalmicroorganism in the generation of a microorganism modified to expressor overexpress a second target enzyme. In turn, the microorganismmodified to express or overexpress a first and a second target enzymecan be modified to express or overexpress a third target enzyme.

Accordingly, a “parental microorganism” functions as a reference cellfor successive genetic modification events. Each modification event canbe accomplished by introducing a nucleic acid molecule in to thereference cell. The introduction facilitates the expression oroverexpression of a target enzyme. It is understood that the term“facilitates” encompasses the activation of endogenous polynucleotidesencoding a target enzyme through genetic modification of e.g., apromoter sequence in a parental microorganism. It is further understoodthat the term “facilitates” encompasses the introduction of heterologouspolynucleotides encoding a target enzyme in to a parental microorganism.

The term “engineer” refers to any manipulation of a microorganism thatresults in a detectable change in the microorganism, wherein themanipulation includes but is not limited to inserting a polynucleotideand/or polypeptide heterologous to the microorganism and mutating apolynucleotide and/or polypeptide native to the microorganism.

The term “mutation” as used herein indicates any modification of anucleic acid and/or polypeptide which results in an altered nucleic acidor polypeptide. Mutations include, for example, point mutations,deletions, or insertions of single or multiple residues in apolynucleotide, which includes alterations arising within aprotein-encoding region of a gene as well as alterations in regionsoutside of a protein-encoding sequence, such as, but not limited to,regulatory or promoter sequences. A genetic alteration may be a mutationof any type. For instance, the mutation may constitute a point mutation,a frame-shift mutation, an insertion, or a deletion of part or all of agene. In addition, in some embodiments of the modified microorganism, aportion of the microorganism genome has been replaced with aheterologous polynucleotide. In some embodiments, the mutations arenaturally-occurring. In other embodiments, the mutations are the resultsof artificial selection pressure. In still other embodiments, themutations in the microorganism genome are the result of geneticengineering.

The term “biosynthetic pathway”, also referred to as “metabolicpathway”, refers to a set of anabolic or catabolic biochemical reactionsfor converting one chemical species into another. Gene products belongto the same “metabolic pathway” if they, in parallel or in series, acton the same substrate, produce the same product, or act on or produce ametabolic intermediate (i.e., metabolite) between the same substrate andmetabolite end product.

The term “heterologous” as used herein with reference to molecules andin particular enzymes and polynucleotides, indicates molecules that areexpressed in an organism other than the organism from which theyoriginated or are found in nature, independently of the level ofexpression that can be lower, equal or higher than the level ofexpression of the molecule in the native microorganism.

On the other hand, the term “native” or “endogenous” as used herein withreference to molecules, and in particular enzymes and polynucleotides,indicates molecules that are expressed in the organism in which theyoriginated or are found in nature, independently of the level ofexpression that can be lower equal or higher than the level ofexpression of the molecule in the native microorganism. It is understoodthat expression of native enzymes or polynucleotides may be modified inrecombinant microorganisms.

The term “feedstock” is defined as a raw material or mixture of rawmaterials supplied to a microorganism or fermentation process from whichother products can be made. For example, a carbon source, such asbiomass or the carbon compounds derived from biomass are a feedstock fora microorganism that produces a biofuel in a fermentation process.However, a feedstock may contain nutrients other than a carbon source.

The term “substrate” or “suitable substrate” refers to any substance orcompound that is converted or meant to be converted into anothercompound by the action of an enzyme. The term includes not only a singlecompound, but also combinations of compounds, such as solutions,mixtures and other materials which contain at least one substrate, orderivatives thereof. Further, the term “substrate” encompasses not onlycompounds that provide a carbon source suitable for use as a startingmaterial, such as any biomass derived sugar, but also intermediate andend product metabolites used in a pathway associated with a recombinantmicroorganism as described herein.

The term “C2-compound” as used as a carbon source for engineered yeastmicroorganisms with mutations in all pyruvate decarboxylase (PDC) genesresulting in a reduction of pyruvate decarboxylase activity of saidgenes refers to organic compounds comprised of two carbon atoms,including but not limited to ethanol and acetate

The term “fermentation” or “fermentation process” is defined as aprocess in which a microorganism is cultivated in a culture mediumcontaining raw materials, such as feedstock and nutrients, wherein themicroorganism converts raw materials, such as a feedstock, intoproducts.

The term “volumetric productivity” or “production rate” is defined asthe amount of product formed per volume of medium per unit of time.Volumetric productivity is reported in gram per liter per hour (g/L/h).

The term “specific productivity” or “specific production rate” isdefined as the amount of product formed per volume of medium per unit oftime per amount of cells. Volumetric productivity is reported in gram ormilligram per liter per hour per OD (g/L/h/OD).

The term “yield” is defined as the amount of product obtained per unitweight of raw material and may be expressed as g product per g substrate(g/g). Yield may be expressed as a percentage of the theoretical yield.“Theoretical yield” is defined as the maximum amount of product that canbe generated per a given amount of substrate as dictated by thestoichiometry of the metabolic pathway used to make the product. Forexample, the theoretical yield for one typical conversion of glucose toisobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose of0.39 g/g would be expressed as 95% of theoretical or 95% theoreticalyield.

The term “titer” is defined as the strength of a solution or theconcentration of a substance in solution. For example, the titer of abiofuel in a fermentation broth is described as g of biofuel in solutionper liter of fermentation broth (g/L).

“Aerobic conditions” are defined as conditions under which the oxygenconcentration in the fermentation medium is sufficiently high for anaerobic or facultative anaerobic microorganism to use as a terminalelectron acceptor.

In contrast, “anaerobic conditions” are defined as conditions underwhich the oxygen concentration in the fermentation medium is too low forthe microorganism to use as a terminal electron acceptor. Anaerobicconditions may be achieved by sparging a fermentation medium with aninert gas such as nitrogen until oxygen is no longer available to themicroorganism as a terminal electron acceptor. Alternatively, anaerobicconditions may be achieved by the microorganism consuming the availableoxygen of the fermentation until oxygen is unavailable to themicroorganism as a terminal electron acceptor.

“Aerobic metabolism” refers to a biochemical process in which oxygen isused as a terminal electron acceptor to make energy, typically in theform of ATP, from carbohydrates. Aerobic metabolism occurs e.g. viaglycolysis and the TCA cycle, wherein a single glucose molecule ismetabolized completely into carbon dioxide in the presence of oxygen.

In contrast, “anaerobic metabolism” refers to a biochemical process inwhich oxygen is not the final acceptor of electrons contained in NADH.Anaerobic metabolism can be divided into anaerobic respiration, in whichcompounds other than oxygen serve as the terminal electron acceptor, andsubstrate level phosphorylation, in which the electrons from NADH areutilized to generate a reduced product via a “fermentative pathway.”

In “fermentative pathways”, NAD(P)H donates its electrons to a moleculeproduced by the same metabolic pathway that produced the electronscarried in NAD(P)H. For example, in one of the fermentative pathways ofcertain yeast strains, NAD(P)H generated through glycolysis transfersits electrons to pyruvate, yielding ethanol. Fermentative pathways areusually active under anaerobic conditions but may also occur underaerobic conditions, under conditions where NADH is not fully oxidizedvia the respiratory chain. For example, above certain glucoseconcentrations, Crabtree positive yeasts produce large amounts ofethanol under aerobic conditions.

The term “byproduct” means an undesired product related to theproduction of a biofuel or biofuel precursor. Byproducts are generallydisposed as waste, adding cost to a production process.

The term “non-fermenting yeast” is a yeast species that fails todemonstrate an anaerobic metabolism in which the electrons from NADH areutilized to generate a reduced product via a fermentative pathway suchas the production of ethanol and CO₂ from glucose. Non-fermentativeyeast can be identified by the “Durham Tube Test” (J. A. Barnett, R. W.Payne, and D. Yarrow. 2000. Yeasts Characteristics and Identification.3^(rd) edition. p. 28-29. Cambridge University Press, Cambridge, UK) orby monitoring the production of fermentation productions such as ethanoland CO₂.

The term “polynucleotide” is used herein interchangeably with the term“nucleic acid” and refers to an organic polymer composed of two or moremonomers including nucleotides, nucleosides or analogs thereof,including but not limited to single stranded or double stranded, senseor antisense deoxyribonucleic acid (DNA) of any length and, whereappropriate, single stranded or double stranded, sense or antisenseribonucleic acid (RNA) of any length, including siRNA. The term“nucleotide” refers to any of several compounds that consist of a riboseor deoxyribose sugar joined to a purine or a pyrimidine base and to aphosphate group, and that are the basic structural units of nucleicacids. The term “nucleoside” refers to a compound (as guanosine oradenosine) that consists of a purine or pyrimidine base combined withdeoxyribose or ribose and is found especially in nucleic acids. The term“nucleotide analog” or “nucleoside analog” refers, respectively, to anucleotide or nucleoside in which one or more individual atoms have beenreplaced with a different atom or with a different functional group.Accordingly, the term polynucleotide includes nucleic acids of anylength, DNA, RNA, analogs and fragments thereof. A polynucleotide ofthree or more nucleotides is also called nucleotidic oligomer oroligonucleotide.

It is understood that the polynucleotides described herein include“genes” and that the nucleic acid molecules described herein include“vectors” or “plasmids.” Accordingly, the term “gene”, also called a“structural gene” refers to a polynucleotide that codes for a particularsequence of amino acids, which comprise all or part of one or moreproteins or enzymes, and may include regulatory (non-transcribed) DNAsequences, such as promoter sequences, which determine for example theconditions under which the gene is expressed. The transcribed region ofthe gene may include untranslated regions, including introns,5′-untranslated region (UTR), and 3′-UTR, as well as the codingsequence.

The term “operon” refers to two or more genes which are transcribed as asingle transcriptional unit from a common promoter. In some embodiments,the genes comprising the operon are contiguous genes. It is understoodthat transcription of an entire operon can be modified (i.e., increased,decreased, or eliminated) by modifying the common promoter.Alternatively, any gene or combination of genes in an operon can bemodified to alter the function or activity of the encoded polypeptide.The modification can result in an increase in the activity of theencoded polypeptide. Further, the modification can impart new activitieson the encoded polypeptide. Exemplary new activities include the use ofalternative substrates and/or the ability to function in alternativeenvironmental conditions.

A “vector” is any means by which a nucleic acid can be propagated and/ortransferred between organisms, cells, or cellular components. Vectorsinclude viruses, bacteriophage, pro-viruses, plasmids, phagemids,transposons, and artificial chromosomes such as YACs (yeast artificialchromosomes), BACs (bacterial artificial chromosomes), and PLACs (plantartificial chromosomes), and the like, that are “episomes,” that is,that replicate autonomously or can integrate into a chromosome of a hostcell. A vector can also be a naked RNA polynucleotide, a naked DNApolynucleotide, a polynucleotide composed of both DNA and RNA within thesame strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugatedDNA or RNA, a liposome-conjugated DNA, or the like, that are notepisomal in nature, or it can be an organism which comprises one or moreof the above polynucleotide constructs such as an agrobacterium or abacterium.

“Transformation” refers to the process by which a vector is introducedinto a host cell. Transformation (or transduction, or transfection), canbe achieved by any one of a number of means including chemicaltransformation (e.g. lithium acetate transformation), electroporation,microinjection, biolistics (or particle bombardment-mediated delivery),or agrobacterium mediated transformation.

The term “enzyme” as used herein refers to any substance that catalyzesor promotes one or more chemical or biochemical reactions, which usuallyincludes enzymes totally or partially composed of a polypeptide, but caninclude enzymes composed of a different molecule includingpolynucleotides.

The term “protein”, “peptide” or “polypeptide” as used herein indicatesan organic polymer composed of two or more amino acidic monomers and/oranalogs thereof. As used herein, the term “amino acid” or “amino acidicmonomer” refers to any natural and/or synthetic amino acids includingglycine and both D or L optical isomers. The term “amino acid analog”refers to an amino acid in which one or more individual atoms have beenreplaced, either with a different atom, or with a different functionalgroup. Accordingly, the term polypeptide includes amino acidic polymerof any length including full length proteins, and peptides as well asanalogs and fragments thereof. A polypeptide of three or more aminoacids is also called a protein oligomer or oligopeptide

The term “homolog”, used with respect to an original enzyme or gene of afirst family or species, refers to distinct enzymes or genes of a secondfamily or species which are determined by functional, structural orgenomic analyses to be an enzyme or gene of the second family or specieswhich corresponds to the original enzyme or gene of the first family orspecies. Most often, homologs will have functional, structural orgenomic similarities. Techniques are known by which homologs of anenzyme or gene can readily be cloned using genetic probes and PCR.Identity of cloned sequences as homolog can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.(Thus, the term “homologous proteins” is defined to mean that the twoproteins have similar amino acid sequences).

The term “analog” or “analogous” refers to nucleic acid or proteinsequences or protein structures that are related to one another infunction only and are not from common descent or do not share a commonancestral sequence. Analogs may differ in sequence but may share asimilar structure, due to convergent evolution. For example, two enzymesare analogs or analogous if the enzymes catalyze the same reaction ofconversion of a substrate to a product, are unrelated in sequence, andirrespective of whether the two enzymes are related in structure.

Cytosolically Localized Isobutanol Pathway Enzymes and RecombinantMicroorganisms Comprising the Same

Biosynthetic pathways for the production of isobutanol and2-methyl-1-butanol by recombinant microorganisms are described by Atsumiet al. (Atsumi et al., 2008, Nature 451: 86-89). One strategy describedherein for improving isobutanol production by recombinant microorganismsis the localization of the enzymes catalyzing the biosyntheticisobutanol pathway to the yeast cytosol. Cytosolic localization of theisobutanol pathway enzymes activity is desirable, especially for theproduction of isobutanol since the ideal biocatalyst (e.g. recombinantmicroorganism) will have the entire isobutanol pathway functionallyexpressed in the same compartment (e.g. preferably in the cytosol). Inaddition, this localization allows the pathway to utilize pyruvate andNAD(P)H that is generated in the cytosol by glycolysis and/or thepentose phosphate pathway without the need for transfer of thesemetabolites to an alternative compartment (i.e. the mitochondria).However, such a strategy of compartmental localization in yeast is notfeasible unless the pathway enzymes exhibit cytosolic activity in thatcompartment. Thus, if one or more of the cytosolically localized pathwayenzymes lacks catalytic activity in the cytosol, high level isobutanolproduction will not occur. As the present application shows in theExamples below, inefficient cytosolic activity of one or or moreisobutanol pathway enzymes (e.g. DHAD or ALS) can limit isobutanolproduction.

The present inventors describe herein cytosolically active isobutanolpathway enzymes and their use in the production of various beneficialmetabolites, such as isobutanol and 2-methyl-1-butanol. Using acombination of genetic selection and biochemical analyses, the presentinventors have identified a number of isobutanol pathway enzymes,including DHAD enzymes, that have activity in the cytosol. Accordingly,in one aspect, the present application describes the discovery of DHADswith enhanced cytosolic activity and shows that these newly identified,cytosolically active DHADs facilitate improved isobutanol productionwhen co-expressed in the cytosol with the remaining four isobutanolpathway enzymes.

As shown in Example 3 below, the native DHAD of yeast is localized tothe mitochondria. Therefore, for economically viable production ofisobutanol to occur in the yeast cytosol, the identification ofheterologous DHAD enzymes that are “cytosolically active” in yeast (i.e.“active in the cytosol” of the yeast) is important. In addition, thepresent application shows that in the absence of ALS, KARI, KIVD, andADH which are “cytosolically active” or “active in the cytosol” in thecytosol of yeast, economically viable isobutanol production will notoccur, thus making identification of native and/or heterologous ALS,KARI, KIVD, and ADH enzymes additionally and/or independently importantto cytosolic isobutanol production.

As used herein, the term “cytosolically active” or “active in thecytosol” means the enzyme exhibits enzymatic activity in the cytosol ofa eukaryotic organism. Cytosolically active enzymes may further beadditionally and/or independently characterized as enzymes thatgenerally exhibit a specific cytosolic activity which is greater thanthe specific mitochondrial activity. In certain respects, a“cytosolically active” enzymes of the present invention exhibit a ratioof the specific activity of the mitochondrial fraction over the specificactivity of the whole cell fraction of less than 1, as determined by themethod disclosed in Example 3 herein. Cytosolically active enzymes mayfurther be additionally and/or independently characterized as enzymesthat, when overexpressed, result in increased activity in the whole cellfraction and do not result in increased activity in the mitochondrialfraction, as determined by the method disclosed in Example 20.Cytosolically active enzymes may further be additionally and/orindependently characterized as enzymes that, when overexpressed as oneof the five enzymes that together comprise the fivestep biosyntheticpathway for the conversion of pyruvate isobutanol, result in increasedisobutanol production compared to enzymes that are not cytosolicallyactive or that are less cytosolically active.

As used herein, the term “cytosolically localized” or “cytosoliclocalization” means the enzyme is localized in the cytosol of aeukaryotic organism. Cytosolically localized enzymes may further beadditionally and/or independently characterized as enzymes that exhibita cytosolic protein level which is greater than the mitochondrialprotein level.

Identification of Cytosolically Active Isobutanol Pathway Enzymes

In one aspect, the present invention encompasses a number of strategiesfor identifying cytosolically active and/or localized isobutanol pathwayenzymes that exhibit cytosolic activity and/or cytosolic localization,as well as methods for modifying said isobutanol pathway enzymes toincrease their ability to exhibit cytosolic activity and/or cytosoliclocalization.

In various embodiments described herein, the isobutanol pathway enzymesmay be derived from a prokaryotic organism. In alternative embodimentsdescribed herein, the isobutanol pathway enzyme may be derived from aeukaryotic organism. In one embodiment, the eukaryotic organism is afungal organism. As described herein, the present inventors have foundthat in general, an enzyme from a fungal source is more likely to showactivity in yeast than a bacterial enzyme expressed in yeast. Inaddition, homologs that are normally expressed in the cytosol aredesired, as a normally cytoplasmic enzyme is likely to show higheractivity in the cytosol as compared to an enzyme that is relocalized tothe cytosol from other organelles, such as the mitochondria. Fungalhomologs of various isobutanol pathway enzymes are often localized tothe mitochondria. The present inventors have found that fungal homologsof isobutanol pathway enzymes that are cytosolically localized willgenerally be expected to exhibit higher activity in the cytosol of yeastthan those of wild-type yeast strains. Thus, in one embodiment, thepresent invention provides fungal isobutanol pathway enzyme homologsthat are cytosolically active and/or cytosolically localized.

Dihydroxyacid Dehydratase (DHAD)

In additional embodiments, at least one of the isobutanol pathwayenzymes exhibiting cytosolic activity is a dihydroxyacid dehydratase(DHAD). In accordance with this embodiment, the present inventionprovides cytosolically active dihydroxyacid dehydratases (DHADs) andfurther describes methods for their use in the production of variousbeneficial metabolites, such as isobutanol and 2-methyl-1-butanol. Asnoted above, biosynthetic pathways for the production of isobutanol and2-methyl-1-butanol have been described (Atsumi et al., 2008, Nature 451:86-89). In these biosynthetic pathways, DHAD catalyzes the conversion of2,3-dihydroxyisovalerate to 2-ketoisovalerate, and2,3-dihydroxy-3-methylvalerate to 2-keto-3-methylvarate, respectively.Using a combination of genetic selection and biochemical analyses, thepresent inventors have identified a number of DHAD homologs that haveactivity in the cytosol.

Among the many strategies for identifying cytosolically active DHADs,the present inventors performed multiway-protein alignments betweenseveral DHAD homologs. Using this analysis, the present inventorsidentified a protein motif that was surprisingly unique to a subset ofDHAD homologs exhibiting cytosolical activity. This protein motif,P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27) was found in DHAD homologsdemonstrating cytosolic activity in yeast. Therefore, in one embodiment,the present invention provides DHAD enzymes comprising the amino acidsequence P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27), wherein X is any naturalor non-natural amino acid, and wherein said DHAD enzyme exhibits theability to convert 2,3-dihydroxyisovalerate to ketoisovalerate in thecytosol. DHAD enzymes harboring this sequence include those derived fromL. lactis (SEQ ID NO: 18), G. forsetii (SEQ ID NO: 17), Acidobacteriabacterium Ellin345 (SEQ ID NO: 16), Saccharopolyspora erythraea (SEQ IDNO: 19), Yarrowia lipolytica (SEQ ID NO: 13), Francisella tularensis(SEQ ID NO: 14), Arabidopsis thaliana (SEQ ID NO: 15), Thermotogapetrophila (SEQ ID NO: 10), and Victivallis vadensis (SEQ ID NO: 11).Also encompassed herein are DHAD enzymes that comprise a motif that isat least about 70% similar, at least about 80% similar, or at leastabout 90% similar to the motif shown in SEQ ID NO: 27.

As described herein, an even more specific version of this motif hasbeen identified by the present inventors. Thus, in a further embodiment,the present invention provides DHAD enzymes comprising the amino acidsequence PIKXXGX(I/L)XIL (SEQ ID NO: 28), wherein X is any natural ornon-natural amino acid, and wherein said DHAD enzyme exhibits theability to convert 2,3-dihydroxyisovalerate to ketoisovalerate in thecytosol. DHAD enzymes harboring this sequence include those derived fromL. lactis (SEQ ID NO: 18), G. forsetii (SEQ ID NO: 17), Acidobacteriabacterium Ellin345 (SEQ ID NO: 16), Y. lipolytica (SEQ ID NO: 13), F.tularensis (SEQ ID NO: 14), A. thaliana (SEQ ID NO: 15), T. petrophila(SEQ ID NO: 10), and V. vadensis (SEQ ID NO: 11). Also encompassedherein are DHAD enzymes that comprise a motif that is at least about 70%similar, at least about 80% similar, or at least about 90% similar tothe motif shown in SEQ ID NO: 28.

As noted above, one such cytosolically active DHAD identified herein isexemplified by the L. lactis DHAD amino acid sequence of SEQ ID NO: 18,which is encoded by the L. lactis ilvD gene. As described herein, thepresent inventors have discovered that yeast strains expressing thecytosolically active L. lactis ilvD (DHAD) exhibit higher isobutanolproduction than yeast strains expressing the S. cerevisiae ILV3 (DHAD),even when the ILV3 from S. cerevisiae is truncated at its N-terminus toremove a putative mitochondrial targeting sequence. In addition to theuse and identification of the cytosolically active DHAD homolog from L.lactis, the present invention encompasses a number of differentstrategies for identifying DHAD enzymes that exhibit cytosolic activityand/or cytosolic localization, as well as methods for modifying DHADs toincrease their ability to exhibit cytosolic activity and/or cytosoliclocalization.

In various embodiments described herein, the DHAD enzymes may be derivedfrom a prokaryotic organism. In one embodiment, the prokaryotic organismis a bacterial organism. In another embodiment, the bacterial organismis L. lactis. In a specific embodiment, the DHAD enzyme from L. lactiscomprises the amino acid sequence of SEQ ID NO: 18. In otherembodiments, the bacterial organisms are of the genus Lactococcus,Gramella, Acidobacteria, Francisella, Thermotoga and Victivallis.

In alternative embodiments, the DHAD enzyme may be derived from aeukaryotic organism. In one embodiment, the eukaryotic organism is afungal organism. In an exemplary embodiment, the fungal organism isNeurospora crassa. In a specific embodiment, the DHAD enzyme from N.crassa comprises the amino acid sequence of SEQ ID NO: 165.

As described herein, the present inventors have found that in general,an enzyme from a fungal source is more likely to show activity in yeastthan a bacterial enzyme expressed in yeast. In addition, homologs thatare normally expressed in the cytosol are desired, as a normallycytoplasmic enzyme is likely to show higher activity in the cytosol ascompared to an enzyme that is relocalized to the cytosol from otherorganelles, such as the mitochondria. Fungal homologs of variousisobutanol pathway enzymes, including DHAD, are often localized to themitochondria. The present inventors have found that fungal homologs ofDHAD that are cytosolically localized will generally be expected toexhibit higher activity in the cytosol of yeast than those of wild-typeyeast strains. Thus, in one embodiment, the present invention providesfungal DHAD homologs that are cytosolically active and/or cytosolicallylocalized.

In another embodiment, the eukaryotic organism is a yeast organism. Inanother embodiment, the eukaryotic organism is selected from the groupconsisting of the genera Enamoeba and Giardia.

In various embodiments described herein, the recombinant microorganismmay exhibit at least about 5 percent greater dihydroxyacid dehydratase(DHAD) activity in the cytosol as compared to the parentalmicroorganism. In another embodiment, the recombinant microorganism mayexhibit at least about 10 percent, at least about 15 percent, aboutleast about 20 percent, at least about 25 percent, at least about 30percent, at least about 35 percent, at least about 40 percent, at leastabout 45 percent, at least about 50 percent, at least about 55 percent,at least about 60 percent, at least about 65 percent, at least about 70percent, at least about 75 percent, at least about 80 percent, at leastabout 100 percent, at least about 200 percent, or at least about 500percent greater dihydroxyacid dehydratase (DHAD) activity in the cytosolas compared to the parental microorganism.

In another embodiment, the present invention provides DHAD enzymes that,when overexpressed in yeast, result in increased activity in the wholecell fraction and do not result in increased activity in themitochondrial fraction. In one embodiment, the DHAD activity in thewhole cell fraction is increased by at least about 2-fold. In anotherembodiment, DHAD activity in the whole cell fraction is increased by atleast about 5-fold. In yet another embodiment, DHAD activity in thewhole cell fraction is increased by at least about 7-fold. In yetanother embodiment, DHAD activity in the whole cell fraction isincreased by at least about 10-fold. In yet another embodiment, DHADactivity in the whole cell fraction is increased by at least about50-fold. In yet another embodiment, DHAD activity in the whole cellfraction is increased by at least about 100-fold.

Acetolactate Synthase (ALS)

As described herein, the isobutanol pathway enzymes in addition to DHADshould preferably be active in the cytosol. These cytosolically activeisobutanol pathway enzymes will generally exhibit enzymatic activity inthe cytosol. For instance, a cytosolically active ALS should generallyexhibit the ability to convert 2 pyruvate to acetolactate in thecytosol. Thus, in various embodiments described herein, at least one ofthe isobutanol pathway enzymes exhibiting cytosolic activity isacetolactate synthase (ALS). In yeasts such as S. cerevisiae, the nativeacetolactate synthase, encoded in S. cerevisiae by the ILV2 gene, isnaturally expressed in the yeast mitochondria. Unlike the endogenousacetolactate synthase of yeast, expression of heterologous, acetolactatesynthases such as the B. subtilis alsS and the L. lactis alsS in yeastoccurs in the yeast cytosol (i.e. cytosolically-localized). Thus,cytosolic expression of acetolactate synthase is achieved bytransforming a yeast with a gene encoding an acetolactate synthaseprotein (EC 2.2.1.6).

ALS homologs that could be cytosolically expressed and localized inyeast are predicted to lack a mitochondrial targeting sequence asanalyzed using mitoprot (Claros et al., 1996, Eur. J. Biochem 241:779-86). Such cytosolically localized ALS proteins can be used as thefirst step in the isobutanol pathway. ALS homologs include, but are notlimited to, the following: the Serratia marcescens ALS (GenBankAccession No. ADH43113.1) (probability of mitochondrial localization0.07), the Enterococcus faecalis ALS (GenBank Accession No.NP_(—)814940) (probability of mitochondrial localization 0.21), theLeuconostoc mesenteroides (GenBank Accession No. YP_(—)818010.1)(probability of mitochondrial localization 0.21), the Staphylococcusaureus ALS (GenBank Accession No. YP_(—)417545) (probability ofmitochondrial localization 0.13), the Burkholderia cenocepacia ALS(GenBank Accession No. YP_(—)624435) (probability of mitochondriallocalization 0.15), the T. atroviride ALS (SEQ ID NO: 71) (probabilityof mitochondrial localization 0.19), the T. stipitatus ALS (SEQ ID NO:72) (probability of mitochondrial localization 0.19), and theMagnaporthe grisea ALS (GenBank Accession No. EDJ99221) (probability ofmitochondrial localization 0.02), a homolog or variant of any of theforegoing, and a polypeptide having at least 60% identity to anyone ofthe foregoing and exhibiting cytosolic ALS activity.

In one embodiment, the cytosolically active ALS is derived from aprokaryotic organism, including, but not limited to B. subtilis or L.lactis, which exhibit cytosolic activity. In another embodiment, the ALSmay be derived from an eukaryotic organism, including, but not limitedto M. grisea, P. nodorum, T. stipitatus, and T. atroviride.

In some embodiments, an ALS enzyme that is predicted to bemitochondrially localized may be mutated or modified to remove or modifyan N-terminal mitochondrial targeting sequence (MTS) to remove oreliminate its ability to target the ALS enzyme to the mitochondria.Removal of the MTS can increase cytosolic localization of the ALS and/orincrease the cytosolic activity of the ALS as compared to the parentalALS.

The conversion of two pyruvate molecules to acetolactate can be carriedout by either an acetohydroxyacid synthase (AHAS) or an acetolactatesynthase (ALS). AHASs are involved in biosynthesis of branched chainamino acids in the mitochondria of yeasts. They are FAD-dependent andare feedback inhibited by branched chain amino acids. ALSs are catabolicand are involved in the conversion of pyruvate to acetoin. ALS areFAD-independent and not feedback inhibited by branched chain aminoacids. In addition, ALSs are specific for the conversion of twopyruvates to acetolactate. Therefore, ALSs are favored over AHASs. Inaddition, in the case of yeast, AHASs are normally mitochondrial,therefore a fungal ALS that is cytoplasmic is favored. Sequence analysishas shown that there is a conserved sequence ‘RFDDR’ found in AHASs thatis not conserved among ALSs (Le et al., 2005, Bull. Korean Chem Soc 26:916-20). This sequence is likely involved in FAD-binding by AHASs andthus could be used to distinguish between the FAD-dependent AHASs andthe FAD-independent ALSs. Using this region to distinguish between AHASsand ALSs BLAST searches of fungal sequence databases were performed andresulted in the identification of ALS homologs from several fungalspecies (M. grisea, P. nodorum, T. atroviride, T. stipitatus, P.marneffei, and Glomerella graminicola). Of these sequences, the ALShomologs from M. grisea, P. nodorum, T. stipitatus, and T. atroviridewill generally be expected to be cytosolically localized.

In one embodiment, the recombinant microorganism may exhibit at leastabout 5 percent greater acetolactate synthase (ALS) activity in thecytosol as compared to the parental microorganism. In anotherembodiment, the recombinant microorganism may exhibit at least about 10percent, at least about 15 percent, about least about 20 percent, atleast about 25 percent, at least about 30 percent, at least about 35percent, at least about 40 percent, at least about 45 percent, at leastabout 50 percent, at least about 55 percent, at least about 60 percent,at least about 65 percent, at least about 70 percent, at least about 75percent, at least about 80 percent, at least about 100 percent, at leastabout 200 percent, or at least about 500 percent greater acetolactatesynthase (ALS) activity in the cytosol as compared to the parentalmicroorganism.

Ketol-Acid Reductoisomerase (KARI)

In additional embodiments, at least one of the isobutanol pathwayenzymes exhibiting cytosolic activity is a ketol-acid reductoisomerase(KARI). A cytosolically active KARI should generally exhibit the abilityto convert acetolactate to 2,3-dihydroxyisovalerate in the cytosol.

In one embodiment, the KARI is derived from a prokaryotic organism,including, but not limited to Escherichia coli, B. subtilis or L.lactis.

in another embodiment, the KARI is derived from a eukaryotic organism,including, but not limited to Piromyces sp. E2, S. cerevisiae, andArabidopsis.Fungal homologs of KARI are generally mitochondriallylocalized. The present inventors have identified a fungal homolog fromthe anaerobic rumenal fungi, Piromyces sp. E2, that is cytosolicallylocalized.

In certain specific embodiments, the KARI comprises an amino acidsequence selected from the group consisting of E. coli (GenBank No:NP_(—)418222, SEQ ID NO: 1), S. cerevisiae (GenBank No: NP_(—)013459,SEQ ID NO: 2), and B. subtilis (GenBank No: CAB14789) and the KARIenzymes from Piromyces sp E2 (GenBank No: CAA76356), B. aphidicola(GenBank No: AAF13807), S. oleracea (GenBank No: CAA40356), O. sativa(GenBank No: NP_(—)001056384, SEQ ID NO: 3), C. reinhardtii (GenBank No:XP_(—)001702649, SEQ ID NO: 6), N. crassa (GenBank No: XP_(—)961335), S.pombe (GenBank No: NP_(—)001018845), L. bicolor (GenBank No:XP_(—)001880867), I. hospitalis (GenBank No: YP_(—)001435197), P.torridus (GenBank No: YP_(—)023851, SEQ ID NO: 7), A. cryptum (GenBankNo: YP_(—)001235669, SEQ ID NO: 5), Cyanobacteria/Synechococcus sp.(GenBank No: YP_(—)473733), Z. mobilis (GenBank No: YP_(—)162876: SEQ IDNO. 8), B. thetaiotaomicron (GenBank No: NP_(—)810987), M. maripaludis(GenBank No: YP_(—)001097443, SEQ ID NO: 4), V. fischeri (GenBank No:YP_(—)205911), Shewanella sp (GenBank No: YP_(—)732498.1), G. forsetti(GenBank No: YP_(—)862142), P. ingrhamaii (GenBank No: YP_(—)942294),and C. hutchinsonii (GenBank No: YP_(—)677763), a homolog or variant ofany of the foregoing, and a polypeptide having at least 60% identity toanyone of the foregoing and exhibiting cytosolic KARI activity.

In additional embodiments, the KARI may be an NADH-dependent KARI. Thus,in one embodiment, the present invention provides recombinantmicroorganisms in which the NADPH-dependent enzymes KARI is replacedwith an enzyme that preferentially depends on NADH (i.e. a KARI that isNADH-dependent). In one embodiment, such enzymes may be identified innature. In an alternative embodiment, such enzymes may be generated byprotein engineering techniques including but not limited to directedevolution or site-directed mutagenesis. NADH-dependent KARIs useful invarious methods of the present invention are described in commonly ownedand co-pending applications U.S. Ser. No. 12/610,784 and PCT/US09/62952(published as WO/2010/051527), which are herein incorporated byreference in their entireties for all purposes.

In one embodiment, a microorganism is provided in which cofactor usageis balanced during the production of a fermentation product and themicroorganism produces the fermentation product at a higher yieldcompared to a modified microorganism in which the cofactor usage in notbalanced. In another embodiment of the present invention, amicroorganism is provided in which the cofactor usage is balanced duringthe production of isobutanol and the microorganism produces isobutanolat a higher yield compared to a modified microorganism in which thecofactor usage in not balanced. Methods for achieving co-factor balanceare described in commonly owned and co-pending applications U.S. Ser.No. 12/610,784 and PCT/US09/62952 (published as WO/2010/051527), whichare herein incorporated by reference in their entireties for allpurposes.

In one embodiment, the recombinant microorganism may exhibit at leastabout 5 percent greater ketol-acid reductoisomerase (KARI) activity inthe cytosol as compared to the parental microorganism. In anotherembodiment, the recombinant microorganism may exhibit at least about 10percent, at least about 15 percent, about least about 20 percent, atleast about 25 percent, at least about 30 percent, at least about 35percent, at least about 40 percent, at least about 45 percent, at leastabout 50 percent, at least about 55 percent, at least about 60 percent,at least about 65 percent, at least about 70 percent, at least about 75percent, at least about 80 percent, at least about 100 percent, at leastabout 200 percent, or at least about 500 percent greater ketol-acidreductoisomerase (KARI) activity in the cytosol as compared to theparental microorganism.

Keto-Acid Decarboxylase (KIVD)

A cytosolically active KIVD should generally exhibit the ability toconvert ketoisovalerate to isobutyraldehyde in the cytosol. In oneembodiment, the cytosolically active KIVD is derived from a prokaryoticorganism, including, but not limited to L. lactis, which exhibitscytosolic activity. In a specific embodiment, the KIVD enzyme from L.lactis comprises the amino acid sequence of SEQ ID NO: 173. Inadditional embodiments, the cytosolically active KIVD is derived from,for example, Enterobacter cloacae (Accession No. P23234.1),Mycobacterium smegmatis (Accession No. A0R480.1), Mycobacteriumtuberculosis (Accession No. O53865.1), Mycobacterium avium (AccessionNo. Q742Q2.1), Azospirillum brasilense (Accession No. P51852.1), B.subtilis (see Oku et al., 1988, J. Biol. Chem. 263: 18386-96), a homologor variant of any of the foregoing, and a polypeptide having at least60% identity to anyone of the foregoing and exhibiting cytosolic KIVDactivity.

In an alternative embodiment, the KIVD may be derived from an eukaryoticorganism.

In one embodiment, the recombinant microorganism may exhibit at leastabout 5 percent greater 2-keto-acid decarboxylase (KIVD) activity in thecytosol as compared to the parental microorganism. In anotherembodiment, the recombinant microorganism may exhibit at least about 10percent, at least about 15 percent, about least about 20 percent, atleast about 25 percent, at least about 30 percent, at least about 35percent, at least about 40 percent, at least about 45 percent, at leastabout 50 percent, at least about 55 percent, at least about 60 percent,at least about 65 percent, at least about 70 percent, at least about 75percent, at least about 80 percent, at least about 100 percent, at leastabout 200 percent, or at least about 500 percent greater 2-keto-aciddecarboxylase (KIVD) activity in the cytosol as compared to the parentalmicroorganism.

Alcohol Dehydroqenase (ADH)

A cytosolically active ADH (used interchangeably herein with isobutanoldehydrogenase, “IDH”) should generally exhibit the ability to convertisobutyraldehyde to isobutanol in the cytosol. In one embodiment, thecytosolically active ADH is derived from a prokaryotic organism,including, but not limited to L. lactis. In a specific embodiment, theADH enzyme from L. lactis comprises the amino acid sequence of SEQ IDNO: 175. In additional embodiments, the ADH is derived from, forexample, Lactobacillus brevis (Accession No. YP_(—)794451.1),Pediococcus acidilactici (Accession No. ZP_(—)06197454.1), Bacilluscereus (Accession No. YP_(—)001374103.1), Bacillus thuringiensis(Accession No. ZP_(—)04101989.1), Leptotrichia goodfellowii (AccessionNo. ZP_(—)06011170.1), Actinobacillus pleuropneumoniae (Accession No.ZP_(—)00134308.2), Streptococcus sanguinis (Accession No.YP_(—)001035842.1), Eikenella corrodens (Accession No.ZP_(—)03713785.1), Exiguobacterium sp. (Accession No.YP_(—)002886170.1), Neisseria elongate (Accession No. ZP_(—)06736067.1),E. coli (Accession No. ZP_(—)06937530.1), Neisseria meningitidis(Accession No. CBA03965.1), Erwinia pyrifoliae (Accession No.CAY75147.1), and Colwellia psychrerythraea (Accession No.YP_(—)270515.1), a homolog or variant of any of the foregoing, and apolypeptide having at least 60% identity to anyone of the foregoing andhaving cytosolic ADH activity.

In an alternative embodiment, the ADH may be derived from an eukaryoticorganism, including, but not limited to S. cerevisiae and D.melanogaster. In a specific embodiment, the ADH enzyme from S.cerevisiae is Adh7. In another specific embodiment, the ADH enzyme fromD. melanogaster comprises the amino acid sequence of SEQ ID NO: 176.

In one embodiment, the recombinant microorganism may exhibit at leastabout 5 percent greater alcohol dehydrogenase (ADH) activity in thecytosol as compared to the parental microorganism. In anotherembodiment, the recombinant microorganism may exhibit at least about 10percent, at least about 15 percent, about least about 20 percent, atleast about 25 percent, at least about 30 percent, at least about 35percent, at least about 40 percent, at least about 45 percent, at leastabout 50 percent, at least about 55 percent, at least about 60 percent,at least about 65 percent, at least about 70 percent, at least about 75percent, at least about 80 percent, at least about 100 percent, at leastabout 200 percent, or at least about 500 percent greater alcoholdehydrogenase (ADH) activity in the cytosol as compared to the parentalmicroorganism.

Chimeric Isobutanol Pathway Enzymes

In another aspect, the present invention provides recombinantmicroorganisms comprising chimeric proteins consisting of isobutanolpathway enzymes. In one embodiment, the chimeric proteins consist of ALSand at least one additional protein. In a specific embodiment, theadditional protein is KARI. In a preferred embodiment, the chimericprotein exhibits the biocatalytic properties of both ALS and KARI. Bycreating a chimeric protein that incorporates the activities of both ALSand KARI, this will generally be expected to reduce the effect ofdiffusion and decreasing the time for spontaneous decomposition tooccur. By using a flexible linker and/or structural and sequenceinformation to create a protein with the biocatalytic properties of bothALS and KARI, this will generally increase the concentration of2-acetolactate at the active site of KARI, causing 2-acetolactate to beconverted to 2,3-dihydroxyisovalerate near its theoretical maximum (verylittle effect of diffusion), and thus, the total concentration of2-acetolactate should remain low correspondingly decreasing itsspontaneous decomposition. This will generally have the effect ofincreasing the rate of conversion of 2-acetolactate to2,3-dihydroxyisovalerate.

In another embodiment, the chimeric proteins consist of KARI and atleast one additional protein. In a specific embodiment, the additionalprotein is DHAD. In a preferred embodiment, the chimeric proteinexhibits the biocatalytic properties of both KARI and DHAD. In each ofthe various embodiments described herein, the proteins may be connectedvia a flexible linker.

Isobutanol Pathway Enzymes Attached to a Protein Scaffold

In another aspect, the present invention provides recombinantmicroorganisms comprising a scaffold system tethered to one or moreisobutanol pathway enzymes. In a specific embodiment, the scaffoldsystem is the MAP kinase scaffold (Ste5) system. In a furtherembodiment, one or more of the isobutanol pathway enzymes may bemodified or mutated to comprise a protein domain allowing for binding tothe scaffold system.

The present inventors have found that via the use of a protein scaffold,the isobutanol pathway enzymes that act in concert as part of a singlepathway can be co-localized. In some embodiments, the scaffold systemsare adapted for binding to the isobutanol pathway enzymes. By tetheringthe enzymes that work together in the pathway to a scaffold protein,they are brought into close physical proximity with each other, thusincreasing the efficiency of the isobutanol production.

There are several advantages to keeping pathway enzymes together on ascaffold system. One is that proteins that normally would localize to anintracellular compartment, like the mitochondria, are partitioned ontothe scaffold, thus keeping a sizeable portion of the protein populationin the cytosol. Another is that the chemical products of each enzyme isphysically close to the next enzyme in the pathway, which speedsreaction time and decreases the possibility that the product would beused in a competing pathway. Finally, unstable products of the enzymeswould be used more quickly, since the next enzyme in the pathway wouldbe adjacent to use it as a substrate, thus decreasing nonproductivedegradation of the product.

In a preferred embodiment, the isobutanol pathway enzymes are arrangedin the sequence in which they are needed to function (i.e. ALS followedby KARI followed by DHAD followed by KIVD followed by ADH). In anotherembodiment, the scaffolded protein complex is targeted to the cytosol byadding localization signals to the scaffold. In yet another embodiment,the scaffolded protein complex is targeted to the cell wall by addinglocalization signals to the scaffold. As would be understood by one ofskill in the art, the scaffold system allows for co-localization ofproteins or enzymes in addition to the isobutanol pathway enzymes. Suchproteins may include chaperone proteins, proteins for the conversion ofxylose to xylulose-5P, cellulases, etc.

Removal and/or Modification of N-Terminal Mitochondrial TargetingSequences

The localization of the enzymes involved in production of isobutanol isdesired to be cytosolic. Cytosolic localization allows for the pathwayto utilize pyruvate and NAD(P)H that is generated in the cytosol byglycolysis and/or the pentose phosphate pathway without the need for thetransfer of these metabolites to an alternative compartment (i.e.mitochondria). However, the yeast enzymes acetohydroxyacid synthase(AHAS; Ilv2+Ilv6), ketol-acid reductoisomerase (KARI; Ilv5), anddihydroxyacid dehydratase (DHAD; Ilv3) that carry out the first threesteps of isobutanol production are physiologically localized to themitochondria. Mitochondrial matrix proteins are typically targeted tothe mitochondria by a N-terminal mitochondrial targeting sequence (MTS),which is then cleaved off in the mitochondria resulting in the ‘mature’form of the enzyme (Paschen et al., 2001, IUBMB Life 52: 101-112).Indeed, the N-terminal targeting sequences for Ilv6 has been defined(Pang et al., 1999 Biochemistry 38: 5222-31). N-terminal deletions ofIlv5 has also been shown to re-localize this enzyme to the cytosol(Omura, 2008, Appl. Microbiol. Biotechnol. 78: 503-513; See also Omura,WO/2009/078108 A1, hereby incorporated by reference in its entirety).

One mechanism identified by the present inventors for the cytosoliclocalization of isobutanol pathway enzymes involves the removal and/ormodification of N-terminal mitochondrial targeting sequences (MTS).Nuclear genome-encoded proteins destined to reside in the mitochondriaoften contain an N-terminal Mitochondrial Targeting Sequence (MTS) thatis recognized by a set of proteins collectively known as mitochondrialimport machinery. Following recognition and import, the MTS is thenphysically cleaved off of the imported protein. In eukaryotes, homologsof two of the isobutanol pathway enzymes, ketol-acid reductoisomerase(KARI, e.g. S. cerevisiae Ilv5) and dihydroxy acid dehydratase (DHAD,e.g. S. cerevisiae Ilv3), are predicted to be mitochondrial, based uponthe presence of an N-terminal MTS as well as several in vivo functionaland mutational studies (See e.g., Omura, F., 2008, Appl Gen & Mol Biot78: 503-513). As described herein, the present inventors have designedisobutanol pathway enzymes, whereby the predicted MTS is removed ormodified. In some instances, there exists experimental evidence for thelength of the MTS. Specifically, the MTS of Ilv6 has been experimentallydefined to be the N-terminal 61 amino acids (Pang et al., 1999,Biochemistry 38: 5222-31). The MTS of Ilv5 has been reported to be theN-terminal 47 residues (Kassow A., 1992, “Metabolic effects of deletingthe region encoding the transit peptide in Saccharomyces cerevisiaeILV5” PhD thesis, University of Copenhagen). In addition, the deletionof the N-terminal 46 amino acids of Ilv5 has been shown to result in anactive enzyme that is localized in the cytosol (Omura, F., 2008, ApplGen & Mol Biot 78: 503-513).

As described herein, the present inventors utilize deletions and/ormodifications of the N-terminal MTS to localize the enzymes of theisobutanol pathway to the cytosol. In various embodiments, the MTS canbe entirely or partly deleted or its sequence can be modified toeliminate its ability to target the protein to the mitochondria. Abenefit of removing the entire MTS is that the resulting protein wouldessentially be the ‘mature’ form of the enzyme. The use of deletion ofthe N-terminal MTS can also be expanded to all enzymes/homologs to beused for isobutanol production. This is especially true for homologsfrom eukaryotic organisms other than S. cerevisiae where the enzymes arelocalized to the mitochondria. In addition, some bacterial homologs mayhave a putative MTS. As bacterial enzymes do not undergo an N-terminalcleavage, N-terminal deletions may be deleterious to these enzymes. Insuch cases, modifications of the sequence to block the MTS function ofthe N-terminal sequence may be preferable as such alterations wouldlikely be less deleterious to the enzyme's activity. N-terminal MTS canbe predicted by MitoProt II (See, e.g., Claros et al., 1996, Eur. J.Biochem. 241: 779-786). Using this program, the lengths of the MTS forIlv2 and Ilv3 were predicted to be the N-terminal 55 and 20 amino acids,respectively. Modification of the MTS as contemplated herein includesthe introduction of one or multiple mutations to inhibit MTS function.It is thought that the mitochondrial import machinery recognizes thealiphatic alpha helix that is formed by the MTS. Thus modifications thatmay inhibit MTS functions would be amino acid changes that would alterthe aliphatic amino acids such as mutating the charged residues. Suchmodification(s) prevent its recognition by the mitochondrial importmachinery and subsequent cleavage of the MTS and import into themitochondria.

Peptide Tags to Augment Cytosolic Localization of Isobutanol PathwayEnzymes

In additional embodiments, the mitochondrially imported isobutanolpathway enzymes can be expressed as a chimeric fusion protein to augmentcytosolic localization. In one embodiment, the isobutanol pathway enzymeis fused to a peptide tag, whereby said isobutanol pathway enzymeexhibits increased cytosolic localization and/or cytosolic activity inyeast as compared to the parental isobutanol pathway enzyme. In oneembodiment, the isobutanol pathway enzyme is fused to a peptide tagfollowing removal of the N-terminal Mitochondrial Targeting Sequence(MTS). In one embodiment, the peptide tag is non-cleavable. In apreferred embodiment, the peptide tag is fused at the N-terminus of theisobutanol pathway enzyme. Peptide tags useful in the present inventionpreferably have the following properties: (1) they do not significantlyhinder the normal enzymatic function of the isobutanol pathway enzyme;(2) it folds in such as a way as to block recognition of an N-terminalMTS by the normal mitochondrial import machinery; (3) it promotes thestable expression and/or folding of the isobutanol pathway enzyme itprecedes; (4) it can be detected, for example, by Western blotting orSDS-PAGE plus Coomassie staining to facilitate analysis of theoverexpressed chimeric protein.

Suitable peptide tags for use in the present invention include, but arenot limited to, ubiquitin, ubiquitin-like (UBL) proteins, myc, HA-tag,green fluorescent protein (GFP), and the maltose binding protein (MBP).Ubiquitin, and the Ubiquitin-like protein (Ubl's) offer severaladvantages. For instance, the use of Ubiquitin or similar Ubl's (e.g.,SUMO) as a solubility- and expression-enhancing fusion partner has beenwell documented (Ecker et al., 1989, J Biol Chem 264: 7715-9;Marblestone et al., 2006, Protein Science 15: 182-9). In fact, in S.cerevisiae, several ribosomal proteins are expressed as C-terminalfusions to ubiquitin. Following translation and protein folding,ubiquitin is cleaved from its co-expressed partner by a highly specificubiquitin hydrolase, which recognizes and requires the extremeC-terminal Gly-Gly motif present in ubiquitin and cleaves immediatelyfollowing this sequence; a similar pathway removes Ubl proteins fromtheir fusion partners.

The invention described here describes a method to re-localize anormally mitochondrial protein or enzyme by expressing it as fusion withan N-terminal, non-cleavable ubiquitin or ubiquitin-like molecule. Indoing so, the re-targeted enzyme enjoys enhanced expression, solubility,and function in the cytosol. In another embodiment, the sequenceencoding the MTS can be replaced with a sequence encoding one or morecopies of the c-myc epitope tag (amino acids EQKLISEEDL, SEQ ID NO: 9),which will generally not target a protein into the mitochondria and caneasily be detected by commercially available antibodies.

Altering the Iron-Sulfur Cluster Domain and/or Redox Active Domain

In general, the yeast cytosol demonstrates a different redox potentialthan a bacterial cell, as well as the yeast mitochondria. As a result,isobutanol pathway enzymes which exhibit an iron sulfur (FeS) domainand/or redox active domain, may require the redox potential of thenative environments to be folded or expressed in a functional form.Expressing some isobutanol pathway enzymes in the yeast cytosol, whichcan harbor unfavorable redox potential, has the propensity to result ininactive proteins, even if the proteins are expressed. The presentinventors have identified a number of different strategies to overcomethis problem, which can arise when an isobutanol pathway enzyme which issuited to a particular environment with a specific redox potential isexpressed in the yeast cytosol.

In one embodiment, the present invention provides isobutanol pathwayenzymes that exhibit a properly folded iron-sulfur cluster domain and/orredox active domain in the cytosol. Such isobutanol pathway enzymes willgenerally comprise a mutated or modified iron-sulfur cluster domainand/or redox active domain, allowing for a non-native isobutanol pathwayenzyme to be expressed in the yeast cytosol in a functional form.

In various embodiments described herein, the recombinant microorganismsmay further comprise a nucleic acid encoding a chaperone protein,wherein said chaperone protein assists the folding of a proteinexhibiting cytosolic activity. In a preferred embodiment, the proteinexhibiting cytosolic activity is DHAD. In one embodiment, the chaperonemay be a native protein. In another embodiment, the chaperone proteinmay be an exogenous protein. In some embodiments, the chaperone proteinmay be selected from the group consisting of: endoplasmic reticulumoxidoreductin 1 (Ero1, Accession No. NP_(—)013576.1), including variantsof Ero1 that have been suitably altered to reduce or prevent its normallocalization to the endoplasmic reticulum; thioredoxins (which includesTrx1, Accession No. NP_(—)013144.1; and Trx2, Accession No.NP_(—)011725.1), thioredoxin reductase (Trr1, Accession No.NP_(—)010640.1); glutaredoxins (which includes Grx1, Accession No.NP_(—)009895.1; Grx2, Accession No. NP_(—)010801.1; Grx3, Accession No.NP_(—)010383.1; Grx4, Accession No. NP_(—)01101.1; Grx5, Accession No.NP_(—)015266.1; Grx6, Accession No. NP_(—)010274.1; Grx7, Accession No.NP_(—)009570.1; Grx8, Accession No. NP_(—)013468.1); glutathionereductase Glr1 (Accession No. NP_(—)015234.1); and Jac1 (Accession No.NP_(—)011497.1), including variants of Jac1 that have been suitablyaltered to reduce or prevent its normal mitochondrial localization; andhomologs or variants thereof.

As described herein, iron-sulfur cluster assembly for insertion intoyeast apo-iron-sulfur proteins begins in yeast mitochondria. To assemblein yeast the active iron-sulfur proteins containing the cluster, eitherthe apo-iron-sulfur protein is imported into the mitochondria from thecytosol and the iron-sulfur cluster is inserted into the protein and theactive protein remains localized in the mitochondria; or the iron-sulfurclusters or precursors thereof are exported from the mitochondria to thecytosol and the active protein is assembled in the cytosol or othercellular compartments.

Targeting of yeast mitochondrial iron-sulfur proteins or non-yeastiron-sulfur proteins to the yeast cytosol can result in such proteinsnot being properly assembled with their iron-sulfur clusters. Thispresent invention overcomes this problem by co-expression and cytosolictargeting in yeast of proteins for iron-sulfur cluster assembly andcluster insertion into apo-iron-sulfur proteins, including iron-sulfurcluster assembly and insertion proteins from organisms other than yeast,together with the apo-iron-sulfur protein to provide assembly of activeiron-sulfur proteins in the yeast cytosol.

Therefore, in one embodiment of this invention, the apo-iron-sulfurprotein DHAD enzyme encoded by the E. coli ilvD gene is expressed inyeast together with E. coli iron-sulfur cluster assembly and insertiongenes comprising either the cyaY, iscS, iscU, iscA, hscB, hscA, fdx andisuX genes or the sufA, sufB, sufC, sufD, sufS and sufE genes. Thisstrategy allows for both the apo-iron-sulfur protein (DHAD) and theiron-sulfur cluster assembly and insertion components (the products ofthe isc or suf genes) to come from the same organism, causing assemblyof the active DHAD iron-sulfur protein in the yeast cytosol. As amodification of this embodiment, for those E. coli iron-sulfur clusterassembly and insertion components that localize to or are predicted tolocalize to the yeast mitochondria upon expression in yeast, the genesfor these components are engineered to eliminate such targeting signalsto ensure localization of the components in the yeast cytoplasm. Thus,in some embodiments, one or more genes encoding an iron-sulfur clusterassembly protein may be mutated or modified to remove a signal peptide,whereby localization of the product of said one or more genes to themitochondria is prevented. In certain embodiments, it may be preferableto overexpress one or more genes encoding an iron-sulfur clusterassembly protein.

In additional embodiments, iron-sulfur cluster assembly and insertioncomponents from other than E. coli can be co-expressed with the E. coliDHAD protein to provide assembly of the active DHAD iron-sulfur clusterprotein. Such iron-sulfur cluster assembly and insertion components fromother organisms can consist of the products of the Helicobacter pylorinifS and nifU genes or the Entamoeba histolytica nifS and nifU genes. Asa modification of this embodiment, for those non-E. coli iron-sulfurcluster assembly and insertion components that localize to or arepredicted to localize to the yeast mitochondria upon expression inyeast, the genes for these components can be engineered to eliminatesuch targeting signals to ensure localization of the components in theyeast cytoplasm.

As a further modification of this embodiment, in addition toco-expression of these proteins in aerobically-grown yeast, theseproteins may be co-expressed in anaerobically-grown yeast to lower theredox state of the yeast cytoplasm to improve assembly of the activeiron-sulfur protein.

In another embodiment, the above iron-sulfur cluster assembly andinsertion components can be co-expressed with DHAD apo-iron-sulfurenzymes other than the E. coli IlvD gene product to generate active DHADenzymes in the yeast cytoplasm. As a modification of this embodiment,for those DHAD enzymes that localize to or are predicted to localize tothe yeast mitochondria upon expression in yeast, then the genes forthese enzymes can be engineered to eliminate such targeting signals toensure localization of the enzymes in the yeast cytoplasm.

In additional embodiments, the above methods used to generate activeDHAD enzymes localized to yeast cytoplasm may be combined with methodsto generate active acetolactate synthase, KARI, KIVD and ADH enzymes inthe same yeast for the production of isobutanol by yeast.

In another embodiment, production of active iron-sulfur proteins otherthan DHAD enzymes in yeast cytoplasm can be accomplished byco-expression with iron-sulfur cluster assembly and insertion proteinsfrom organisms other than yeast, with proper targeting of the proteinsto the yeast cytoplasm if necessary and expression in anaerobicallygrowing yeast if needed to improve assembly of the active proteins.

In another embodiment, the iron-sulfur cluster assembly protein encodinggenes may be derived from eukaryotic organisms, including, but notlimited to yeasts and plants. In one embodiment, the iron-sulfur clusterprotein encoding genes are derived from a yeast organism, including, butnot limited to S. cerevisiae. In specific embodiments, the yeast derivedgenes encoding iron-sulfur cluster assembly proteins are selected fromthe group consisting of Cfd1 (Accession No. NP_(—)012263.1), Nbp35(Accession No. NP_(—)011424.1), Nar1 (Accession No. NP_(—)014159.1),Cia1 (Accession No. NP_(—)010553.1), and homologs or variants thereof.In a further embodiment, the iron-sulfur cluster assembly proteinencoding genes may be derived from plant nuclear genes which encodeproteins translocated to chloroplast or plant genes found in thechloroplast genome itself.

As noted above, the iron-sulfur cluster assembly genes may be derivedfrom eukaryotic organisms, including, but not limited to yeasts andplants. In one embodiment, the iron-sulfur cluster genes are derivedfrom a yeast organism, including, but not limited to S. cerevisiae. Inspecific embodiments, the yeast derived iron-sulfur cluster assemblygenes are selected from the group consisting of CFD1, NBP35, NAR1, CIA1,and homologs or variants thereof. In a further embodiment, theiron-sulfur cluster assembly genes may be derived from a plantchloroplast.

In certain embodiments described herein, it may be desirable to reduceor eliminate the activity and/or proteins levels of one or moreiron-sulfur cluster containing cytosolic proteins. This modificationincreases the capacity of a yeast to incorporate [Fe—S] clusters intocytosolically expressed proteins wherein said proteins can be nativeproteins that are expressed in a non-native compartment or heterologousproteins. This is achieved by deletion of a highly expressed nativecytoplasmic [Fe—S]-dependent protein. More specifically, the gene LEU1is deleted coding for the 3-isopropylmalate dehydratase which catalysesthe conversion of 3-isopropylmalate into 2-isopropylmalate as part ofthe leucine biosynthetic pathway in yeast. Leu1p contains an 4Fe-4Scluster which takes part in the catalysis of the dehydratase. DHAD alsocontains a 4Fe-4S cluster involved in its dehydratase activity.Therefore, although the two enzymes have different substrate preferencesthe process of incorporation of the Fe—S cluster is generally similarfor the two proteins. Given that Leu1p is present in yeast at 10000molecules per cell (Ghaemmaghami et al., 2003, Nature 425: 737),deletion of LEU1 therefore ensures that the cell has enough sparecapacity to incorporate [Fe—S] clusters into at least 10000 molecules ofcytosolically expressed DHAD. Taking into account the specific activityof DHAD (E. coli DHAD is reported to have a specific activity of 63U/mg) (Flint et al., 1993, J Biological Chem 268: 14732), the LEU1deletion yeast strain would generally exhibit an increased capacity forDHAD activity in the cytosol as measured in cell lysate.

In alternative embodiments, it may be desirable to further overexpressan additional enzyme that converts 2,3-dihydroxyisovalerate toketoisovalerate in the cytosol. In a specific embodiment, the enzyme maybe selected from the group consisting of 3-isopropylmalate dehydratase(Leu1p) and imidazoleglycerol-phosphate dehydrogenase (His3p). BecauseDHAD activity is limited in the cytosol, alternative dehydratases thatconvert dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) and arephysiologically localized to the yeast cytosol may be utilized. Leu1pand His3p are dehydratases that potentially may exhibit affinity forDHIV. Leu1p is an Fe—S binding protein that is involved in leucinebiosynthesis and is also normally localized to the cytosol. His3p isinvolved in histidine biosynthesis and is similar to Leu1p, it isgenerally localized to the cytosol or predicted to be localized to thecytosol. This modification overcomes the problem of a DHAD that islimiting isobutanol production in the cytosol of yeast. The use of analternative dehydratase that has activity in the cytosol with a lowactivity towards DHIV may thus be used in place of the DHAD in theisobutanol pathway. As described herein, such enzyme may be furtherengineered to increase activity with DHIV.

The Microorganism in General

Native producers of 1-butanol, such as Clostridium acetobutylicum, areknown, but these organisms also generate byproducts such as acetone,ethanol, and butyrate during fermentations. Furthermore, thesemicroorganisms are relatively difficult to manipulate, withsignificantly fewer tools available than in more commonly usedproduction hosts such as S. cerevisiae or E. coli. Additionally, thephysiology and metabolic regulation of these native producers are muchless well understood, impeding rapid progress towards high-efficiencyproduction. Furthermore, no native microorganisms have been identifiedthat can metabolize glucose into isobutanol in industrially relevantquantities.

The production of isobutanol and other fusel alcohols by various yeastspecies, including Saccharomyces cerevisiae is of special interest tothe distillers of alcoholic beverages, for whom fusel alcoholsconstitute often undesirable off-notes. Production of isobutanol inwild-type yeasts has been documented on various growth media, rangingfrom grape must from winemaking (Romano et al., 2003, World J. ofMicrobiol Biot. 19: 311-5), in which 12-219 mg/L isobutanol wereproduced, to supplemented minimal media (Oliviera et al., 2005, World J.of Microbiol Blot. 21: 1569-76), producing 16-34 mg/L isobutanol. Workfrom Dickinson et al. (J Biol Chem. 272: 26871-8, 1997) has identifiedthe enzymatic steps utilized in an endogenous S. cerevisiae pathwayconverting branch-chain amino acids (e.g., valine or leucine) toisobutanol.

Recombinant microorganisms provided herein can express a plurality ofheterologous and/or native target enzymes involved in pathways for theproduction of isobutanol from a suitable carbon source.

Accordingly, “engineered” or “modified” microorganisms are produced viathe introduction of genetic material into a host or parentalmicroorganism of choice and/or by modification of the expression ofnative genes, thereby modifying or altering the cellular physiology andbiochemistry of the microorganism. Through the introduction of geneticmaterial and/or the modification of the expression of native genes theparental microorganism acquires new properties, e.g. the ability toproduce a new, or greater quantities of, an intracellular metabolite. Asdescribed herein, the introduction of genetic material into and/or themodification of the expression of native genes in a parentalmicroorganism results in a new or modified ability to produceisobutanol. The genetic material introduced into and/or the genesmodified for expression in the parental microorganism contains gene(s),or parts of genes, coding for one or more of the enzymes involved in abiosynthetic pathway for the production of isobutanol and may alsoinclude additional elements for the expression and/or regulation ofexpression of these genes, e.g. promoter sequences.

In addition to the introduction of a genetic material into a host orparental microorganism, an engineered or modified microorganism can alsoinclude alteration, disruption, deletion or knocking-out of a gene orpolynucleotide to alter the cellular physiology and biochemistry of themicroorganism. Through the alteration, disruption, deletion orknocking-out of a gene or polynucleotide the microorganism acquires newor improved properties (e.g., the ability to produce a new metabolite orgreater quantities of an intracellular metabolite, improve the flux of ametabolite down a desired pathway, and/or reduce the production ofbyproducts).

Recombinant microorganisms provided herein may also produce metabolitesin quantities not available in the parental microorganism. A“metabolite” refers to any substance produced by metabolism or asubstance necessary for or taking part in a particular metabolicprocess. A metabolite can be an organic compound that is a startingmaterial (e.g., glucose or pyruvate), an intermediate (e.g.,2-ketoisovalerate), or an end product (e.g., isobutanol) of metabolism.Metabolites can be used to construct more complex molecules, or they canbe broken down into simpler ones. Intermediate metabolites may besynthesized from other metabolites, perhaps used to make more complexsubstances, or broken down into simpler compounds, often with therelease of chemical energy.

Exemplary metabolites include glucose, pyruvate, and isobutanol. Themetabolite isobutanol can be produced by a recombinant microorganismwhich expresses or over-expresses a metabolic pathway that convertspyruvate to isobutanol. An exemplary metabolic pathway that convertspyruvate to isobutanol may be comprised of an acetohydroxy acid synthase(ALS), a ketolacid reductoisomerase (KARI), a dihyroxy-acid dehydratase(DHAD), a 2-keto-acid decarboxylase (KIVD), and an alcohol dehydrogenase(ADH).

Accordingly, provided herein are recombinant microorganisms that produceisobutanol and in some aspects may include the elevated expression oftarget enzymes such as ALS, KARI, DHAD, KIVD, and ADH

The disclosure identifies specific genes useful in the methods,compositions and organisms of the disclosure; however it will berecognized that absolute identity to such genes is not necessary. Forexample, changes in a particular gene or polynucleotide comprising asequence encoding a polypeptide or enzyme can be performed and screenedfor activity. Typically such changes comprise conservative mutation andsilent mutations. Such modified or mutated polynucleotides andpolypeptides can be screened for expression of a functional enzyme usingmethods known in the art.

Due to the inherent degeneracy of the genetic code, otherpolynucleotides which encode substantially the same or functionallyequivalent polypeptides can also be used to clone and express thepolynucleotides encoding such enzymes.

As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms typically use a subset of these codons. The codonsthat are utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Codons can be substituted to reflect the preferred codon usageof the host, a process sometimes called “codon optimization” or“controlling for species codon bias.”

Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (Murray et al., 1989, Nucl Acids Res. 17:477-508) can be prepared, for example, to increase the rate oftranslation or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, as compared with transcriptsproduced from a non-optimized sequence. Translation stop codons can alsobe modified to reflect host preference. For example, typical stop codonsfor S. cerevisiae and mammals are UAA and UGA, respectively. The typicalstop codon for monocotyledonous plants is UGA, whereas insects and E.coli commonly use UAA as the stop codon (Dalphin et al., 1996, NuclAcids Res. 24: 216-8). Methodology for optimizing a nucleotide sequencefor expression in a plant is provided, for example, in U.S. Pat. No.6,015,891, and the references cited therein.

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of DNA compounds differing intheir nucleotide sequences can be used to encode a given enzyme of thedisclosure. The native DNA sequence encoding the biosynthetic enzymesdescribed above are referenced herein merely to illustrate an embodimentof the disclosure, and the disclosure includes DNA compounds of anysequence that encode the amino acid sequences of the polypeptides andproteins of the enzymes utilized in the methods of the disclosure. Insimilar fashion, a polypeptide can typically tolerate one or more aminoacid substitutions, deletions, and insertions in its amino acid sequencewithout loss or significant loss of a desired activity. The disclosureincludes such polypeptides with different amino acid sequences than thespecific proteins described herein so long as they modified or variantpolypeptides have the enzymatic anabolic or catabolic activity of thereference polypeptide. Furthermore, the amino acid sequences encoded bythe DNA sequences shown herein merely illustrate embodiments of thedisclosure.

In addition, homologs of enzymes useful for generating metabolites areencompassed by the microorganisms and methods provided herein.

As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences have at leastabout 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percentidentity of two amino acid sequences, or of two nucleic acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). In one embodiment, thelength of a reference sequence aligned for comparison purposes is atleast 30%, typically at least 40%, more typically at least 50%, evenmore typically at least 60%, and even more typically at least 70%, 80%,90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (See,e.g., Pearson W. R., 1994, Methods in Mol Biol 25: 365-89.

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See commonly owned and co-pending application US 2009/0226991.A typical algorithm used comparing a molecule sequence to a databasecontaining a large number of sequences from different organisms is thecomputer program BLAST. When searching a database containing sequencesfrom a large number of different organisms, it is typical to compareamino acid sequences. Database searching using amino acid sequences canbe measured by algorithms described in commonly owned and co-pendingapplication US 2009/0226991.

The disclosure provides recombinant microorganisms comprising abiochemical pathway for the production of isobutanol from a suitablesubstrate at a high yield. A recombinant microorganism of the disclosurecomprises one or more recombinant polynucleotides within the genome ofthe organism or external to the genome within the organism. Themicroorganism can comprise a reduction in expression, disruption orknockout of a gene found in the wild-type organism and/or introductionof a heterologous polynucleotide and/or expression or overexpression ofan endogenous polynucleotide.

In one aspect, the disclosure provides a recombinant microorganismcomprising elevated expression of at least one target enzyme as comparedto a parental microorganism or encodes an enzyme not found in theparental organism. In another or further aspect, the microorganismcomprises a reduction in expression, disruption or knockout of at leastone gene encoding an enzyme that competes with a metabolite necessaryfor the production of isobutanol. The recombinant microorganism producesat least one metabolite involved in a biosynthetic pathway for theproduction of isobutanol. In general, the recombinant microorganismscomprises at least one recombinant metabolic pathway that comprises atarget enzyme and may further include a reduction in activity orexpression of an enzyme in a competitive biosynthetic pathway. Thepathway acts to modify a substrate or metabolic intermediate in theproduction of isobutanol. The target enzyme is encoded by, and expressedfrom, a polynucleotide derived from a suitable biological source. Insome embodiments, the polynucleotide comprises a gene derived from aprokaryotic or eukaryotic source and recombinantly engineered into themicroorganism of the disclosure. In other embodiments, thepolynucleotide comprises a gene that is native to the host organism.

It is understood that a range of microorganisms can be modified toinclude a recombinant metabolic pathway suitable for the production ofisobutanol. In various embodiments, microorganisms may be selected fromyeast microorganisms. Yeast microorganisms for the production ofisobutanol may be selected based on certain characteristics:

One characteristic may include the property that the microorganism isselected to convert various carbon sources into isobutanol. The term“carbon source” generally refers to a substance suitable to be used as asource of carbon for prokaryotic or eukaryotic cell growth. Examples ofsuitable carbon sources are described in commonly owned and co-pendingapplication US 2009/0226991. Accordingly, in one embodiment, therecombinant microorganism herein disclosed can convert a variety ofcarbon sources to products, including but not limited to glucose,galactose, mannose, xylose, arabinose, lactose, sucrose, and mixturesthereof.

The recombinant microorganism may thus further include a pathway for thefermentation of isobutanol from five-carbon (pentose) sugars includingxylose. Most yeast species metabolize xylose via a complex route, inwhich xylose is first reduced to xylitol via a xylose reductase (XR)enzyme. The xylitol is then oxidized to xylulose via a xylitoldehydrogenase (XDH) enzyme. The xylulose is then phosphorylated via axylulokinase (XK) enzyme. This pathway operates inefficiently in yeastspecies because it introduces a redox imbalance in the cell. Thexylose-to-xylitol step uses NADH as a cofactor, whereas thexylitol-to-xylulose step uses NADPH as a cofactor. Other processes mustoperate to restore the redox imbalance within the cell. This often meansthat the organism cannot grow anaerobically on xylose or other pentosesugar. Accordingly, a yeast species that can efficiently ferment xyloseand other pentose sugars into a desired fermentation product istherefore very desirable.

Thus, in one aspect, the recombinant is engineered to express afunctional exogenous xylose isomerase. Exogenous xylose isomerasesfunctional in yeast are known in the art. See, e.g., Rajgarhia et al,US20060234364, which is herein incorporated by reference in itsentirety. In an embodiment according to this aspect, the exogenousxylose isomerase gene is operatively linked to promoter and terminatorsequences that are functional in the yeast cell. In a preferredembodiment, the recombinant microorganism further has a deletion ordisruption of a native gene that encodes for an enzyme (e.g. XR and/orXDH) that catalyzes the conversion of xylose to xylitol. In a furtherpreferred embodiment, the recombinant microorganism also contains afunctional, exogenous xylulokinase (XK) gene operatively linked topromoter and terminator sequences that are functional in the yeast cell.In one embodiment, the xylulokinase (XK) gene is overexpressed.

In one embodiment, the microorganism has reduced or no pyruvatedecarboxylase (PDC) activity. PDC catalyzes the decarboxylation ofpyruvate to acetaldehyde, which is then reduced to ethanol by ADH via anoxidation of NADH to NADH+. Ethanol production is the main pathway tooxidize the NADH from glycolysis. Deletion of this pathway increases thepyruvate and the reducing equivalents (NADH) available for theisobutanol pathway. Accordingly, deletion of PDC genes can furtherincrease the yield of isobutanol.

In another embodiment, the microorganism has reduced or noglycerol-3-phosphate dehydrogenase (GPD) activity. GPD catalyzes thereduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate(G3P) via the oxidation of NADH to NAD+. Glycerol is then produced fromG3P by Glycerol-3-phosphatase (GPP). Glycerol production is a secondarypathway to oxidize excess NADH from glycolysis. Reduction or eliminationof this pathway would increase the pyruvate and reducing equivalents(NADH) available for the isobutanol pathway. Thus, deletion of GPD genescan further increase the yield of isobutanol.

In yet another embodiment, the microorganism has reduced or no PDCactivity and reduced or no GPD activity.

In one embodiment, the yeast microorganisms may be selected from the“Saccharomyces Yeast Clade”, as described in commonly owned andco-pending application US 2009/0226991.

The term “Saccharomyces sensu stricto” taxonomy group is a cluster ofyeast species that are highly related to S. cerevisiae (Rainieri et al.,2003, J. Biosci Bioengin 96: 1-9). Saccharomyces sensu stricto yeastspecies include but are not limited to S. cerevisiae, S. cerevisiae, S.kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis andhybrids derived from these species (Masneuf et al., 1998, Yeast 7:61-72).

An ancient whole genome duplication (WGD) event occurred during theevolution of the hemiascomycete yeast and was discovered usingcomparative genomic tools (Kellis et al., 2004, Nature 428: 617-24;Dujon et al., 2004, Nature 430:35-44; Langkjaer et al., 2003, Nature428: 848-52; Wolfe et al., 1997, Nature 387: 708-13). Using this majorevolutionary event, yeast can be divided into species that diverged froma common ancestor following the WGD event (termed “post-WGD yeast”herein) and species that diverged from the yeast lineage prior to theWGD event (termed “pre-WGD yeast” herein).

Accordingly, in one embodiment, the yeast microorganism may be selectedfrom a post-WGD yeast genus, including but not limited to Saccharomycesand Candida. The favored post-WGD yeast species include: S. cerevisiae,S. uvarum, S. bayanus, S. paradoxus, S. castelli, and C. glabrata.

In another embodiment, the yeast microorganism may be selected from apre-whole genome duplication (pre-WGD) yeast genus including but notlimited to Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia,Debaryomyces, Hansenula, Yarrowia and, Schizosaccharomyces.Representative pre-WGD yeast species include: S. kluyveri, K.thermotolerans, K. marxianus, K. waltii, K. lactis, C. tropicalis, P.pastoris, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I.scutulata, D. hansenii, H. anomala, Y. lipolytica, and S. pombe.

A yeast microorganism may be either Crabtree-negative orCrabtree-positive as described in described in commonly owned andco-pending application US 2009/0226991. In one embodiment the yeastmicroorganism may be selected from yeast with a Crabtree-negativephenotype including but not limited to the following genera:Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida.Crabtree-negative species include but are not limited to: K. lactis, K.marxianus, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I.scutulata, H. anomala, and C. utilis. In another embodiment, the yeastmicroorganism may be selected from a yeast with a Crabtree-positivephenotype, including but not limited to Saccharomyces, Kluyveromyces,Zygosaccharomyces, Debaryomyces, Pichia and Schizosaccharomyces.Crabtree-positive yeast species include but are not limited to: S.cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, S.kluyveri, K. thermotolerans, C. glabrata, Z. bailli, Z. rouxii, D.hansenii, P. pastorius, and S. pombe.

Another characteristic may include the property that the microorganismis that it is non-fermenting. In other words, it cannot metabolize acarbon source anaerobically while the yeast is able to metabolize acarbon source in the presence of oxygen. Nonfermenting yeast refers toboth naturally occurring yeasts as well as genetically modified yeast.During anaerobic fermentation with fermentative yeast, the main pathwayto oxidize the NADH from glycolysis is through the production ofethanol. Ethanol is produced by alcohol dehydrogenase (ADH) via thereduction of acetaldehyde, which is generated from pyruvate by pyruvatedecarboxylase (PDC). In one embodiment, a fermentative yeast can beengineered to be non-fermentative by the reduction or elimination of thenative PDC activity. Thus, most of the pyruvate produced by glycolysisis not consumed by PDC and is available for the isobutanol pathway.Deletion of this pathway increases the pyruvate and the reducingequivalents available for the isobutanol pathway. Fermentative pathwayscontribute to low yield and low productivity of isobutanol. Accordingly,deletion of PDC may increase yield and productivity of isobutanol.

In some embodiments, the recombinant microorganisms may bemicroorganisms that are non-fermenting yeast microorganisms, including,but not limited to those, classified into a genera selected from thegroup consisting of Tricosporon, Rhodotorula, or Myxozyma.

In one embodiment, a yeast microorganism is engineered to convert acarbon source, such as glucose, to pyruvate by glycolysis and thepyruvate is converted to isobutanol via an engineered isobutanol pathway(See, e.g., WO/2007/050671, WO/2008/098227, and Atsumi et al., 2008,Nature 45: 86-9). Alternative pathways for the production of isobutanolhave been described in WO/2007/050671 and in Dickinson et al., 1998, JBiol Chem 273:25751-6.

Accordingly, the engineered isobutanol pathway to convert pyruvate toisobutanol can be comprised of the following reactions:

1. 2 pyruvate→acetolactate+CO₂

2. acetolactate+NAD(P)H→2,3-dihydroxyisovalerate+NAD(P)⁺

3. 2,3-dihydroxyisovalerate→alpha-ketoisovalerate

4. alpha-ketoisovalerate→isobutyraldehyde+CO₂

5. isobutyraldehyde+NAD(P)H→isobutanol+NAD(P)

These reactions are carried out by the enzymes 1) Acetolactate Synthase(ALS), 2) Keto-acid Reducto-Isomerase (KARI), 3) Dihydroxy-aciddehydratase (DHAD), 4) Keto-isovalerate decarboxylase (KIVD), and 5) anAlcohol dehydrogenase (ADH) (FIG. 1). In another embodiment, the yeastmicroorganism is engineered to overexpress these enzymes. For example,these enzymes can be encoded by native genes. Alternatively, theseenzymes can be encoded by heterologous genes. For example, ALS can beencoded by the alsS gene of B. subtilis, alsS of L. lactis, or the ilvKgene of K. pneumonia. For example, KARI can be encoded by the ilvC genesof E. coli, C. glutamicum, M. maripaludis, or Piromyces sp E2. Forexample, DHAD can be encoded by the ilvD genes of E. coli, C.glutamicum, or L. lactis. For example, KIVD can be encoded by the kivDgene of L. lactis. ADH can be encoded by ADH2, ADH6, or ADH7 of S.cerevisiae.

In one embodiment, pathway steps 2 and 5 may be carried out by KARI andADH enzymes that utilize NADH (rather than NADPH) as a co-factor. Suchenzymes are described in commonly owned and co-pending applications U.S.Ser. No. 12/610,784 and PCT/US09/62952 (published as WO/2010/051527),which are herein incorporated by reference in their entireties for allpurposes. The present inventors have found that utilization ofNADH-dependent KARI and ADH enzymes to catalyze pathway steps 2 and 5,respectively, surprisingly enables production of isobutanol underanaerobic conditions. Thus, in one embodiment, the recombinantmicroorganisms of the present invention may use an NADH-dependent KARIto catalyze the conversion of acetolactate (+NADH) to produce2,3-dihydroxyisovalerate. In another embodiment, the recombinantmicroorganisms of the present invention may use an NADH-dependent ADH tocatalyze the conversion of isobutyraldehyde (+NADH) to produceisobutanol. In yet another embodiment, the recombinant microorganisms ofthe present invention may use both an NADH-dependent KARI to catalyzethe conversion of acetolactate (+NADH) to produce2,3-dihydroxyisovalerate, and an NADH-dependent ADH to catalyze theconversion of isobutyraldehyde (+NADH) to produce isobutanol.

The yeast microorganism of the invention may be engineered to haveincreased ability to convert pyruvate to isobutanol. In one embodiment,the yeast microorganism may be engineered to have increased ability toconvert pyruvate to isobutyraldehyde. In another embodiment, the yeastmicroorganism may be engineered to have increased ability to convertpyruvate to keto-isovalerate. In another embodiment, the yeastmicroorganism may be engineered to have increased ability to convertpyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeastmicroorganism may be engineered to have increased ability to convertpyruvate to acetolactate.

Furthermore, any of the genes encoding the foregoing enzymes (or anyothers mentioned herein (or any of the regulatory elements that controlor modulate expression thereof)) may be optimized by genetic/proteinengineering techniques, such as directed evolution or rationalmutagenesis, which are known to those of ordinary skill in the art. Suchaction allows those of ordinary skill in the art to optimize the enzymesfor expression and activity in yeast.

In addition, genes encoding these enzymes can be identified from otherfungal and bacterial species and can be expressed for the modulation ofthis pathway. A variety of organisms could serve as sources for theseenzymes, including, but not limited to, Saccharomyces spp., including S.cerevisiae and S. uvarum, Kluyveromyces spp., including K.thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenulaspp., including H. polymorpha, Candida spp., Trichosporon spp.,Yamadazyma spp., including Y. spp. stipitis, Torulaspora pretoriensis,Schizosaccharomyces spp., including S. pombe, Cryptococcus spp.,Aspergillus spp., Neurospora spp., or Ustilago spp. Sources of genesfrom anaerobic fungi include, but not limited to, Piromyces spp.,Orpinomyces spp., or Neocallimastix spp. Sources of prokaryotic enzymesthat are useful include, but not limited to, Escherichia. coli,Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridiumspp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp.,Enterobacter spp., and Salmonella spp.

Methods in General Identification of PDC and GPD in a YeastMicroorganism

Any method can be used to identify genes that encode for enzymes withpyruvate decarboxylase (PDC) activity or glycerol-3-phosphatedehydrogenase (GPD) activity. Suitable methods for the identification ofPDC and GPD are described in co-pending applications U.S. Ser. No.12/343,375 (published as US 2009/0226991), U.S. Ser. No. 12/696,645, andU.S. Ser. No. 12/820,505, which claim priority to U.S. ProvisionalApplication 61/016,483, all of which are herein incorporated byreference in their entireties for all purposes.

Genetic Insertions and Deletions

Any method can be used to introduce a nucleic acid molecule into yeastand many such methods are well known. For example, transformation andelectroporation are common methods for introducing nucleic acid intoyeast cells. See, e.g., Gietz et al., 1992, Nuc Acids Res. 27: 69-74;Ito et al., 1983, J. Bacteriol. 153: 163-8; and Becker et al., 1991,Methods in Enzymology 194: 182-7.

In an embodiment, the integration of a gene of interest into a DNAfragment or target gene of a yeast microorganism occurs according to theprinciple of homologous recombination. According to this embodiment, anintegration cassette containing a module comprising at least one yeastmarker gene and/or the gene to be integrated (internal module) isflanked on either side by DNA fragments homologous to those of the endsof the targeted integration site (recombinogenic sequences). Aftertransforming the yeast with the cassette by appropriate methods, ahomologous recombination between the recombinogenic sequences may resultin the internal module replacing the chromosomal region in between thetwo sites of the genome corresponding to the recombinogenic sequences ofthe integration cassette. (Orr-Weaver et al., 1981, PNAS USA 78:6354-58).

In an embodiment, the integration cassette for integration of a gene ofinterest into a yeast microorganism includes the heterologous gene underthe control of an appropriate promoter and terminator together with theselectable marker flanked by recombinogenic sequences for integration ofa heterologous gene into the yeast chromosome. In an embodiment, theheterologous gene includes an appropriate native gene desired toincrease the copy number of a native gene(s). The selectable marker genecan be any marker gene used in yeast, including but not limited to,HIS3, TRP1, LEU2, URA3, bar, ble, hph, and kan. The recombinogenicsequences can be chosen at will, depending on the desired integrationsite suitable for the desired application.

In another embodiment, integration of a gene into the chromosome of theyeast microorganism may occur via random integration (Kooistra et al.,2004, Yeast 21: 781-792).

Additionally, in an embodiment, certain introduced marker genes areremoved from the genome using techniques well known to those skilled inthe art. For example, URA3 marker loss can be obtained by plating URA3containing cells in FOA (5-fluoro-orotic acid) containing medium andselecting for FOA resistant colonies (Boeke et al., 1984, Mol. Gen.Genet 197: 345-47).

The exogenous nucleic acid molecule contained within a yeast cell of thedisclosure can be maintained within that cell in any form. For example,exogenous nucleic acid molecules can be integrated into the genome ofthe cell or maintained in an episomal state that can stably be passed on(“inherited”) to daughter cells. Such extra-chromosomal genetic elements(such as plasmids, etc.) can additionally contain selection markers thatensure the presence of such genetic elements in daughter cells.Moreover, the yeast cells can be stably or transiently transformed. Inaddition, the yeast cells described herein can contain a single copy, ormultiple copies of a particular exogenous nucleic acid molecule asdescribed above.

Reduction of Enzymatic Activity

Yeast microorganisms within the scope of the invention may have reducedenzymatic activity such as reduced glycerol-3-phosphate dehydrogenaseactivity. The term “reduced” as used herein with respect to a particularenzymatic activity refers to a lower level of enzymatic activity thanthat measured in a comparable yeast cell of the same species. The termreduced also refers to the elimination of enzymatic activity than thatmeasured in a comparable yeast cell of the same species. Thus, yeastcells lacking glycerol-3-phosphate dehydrogenase activity are consideredto have reduced glycerol-3-phosphate dehydrogenase activity since most,if not all, comparable yeast strains have at least someglycerol-3-phosphate dehydrogenase activity. Such reduced enzymaticactivities can be the result of lower enzyme concentration, lowerspecific activity of an enzyme, or a combination thereof. Many differentmethods can be used to make yeast having reduced enzymatic activity. Forexample, a yeast cell can be engineered to have a disruptedenzyme-encoding locus using common mutagenesis or knock-out technology.In addition, certain point-mutation(s) can be introduced which resultsin an enzyme with reduced activity.

Alternatively, antisense technology can be used to reduce enzymaticactivity. For example, yeast can be engineered to contain a cDNA thatencodes an antisense molecule that prevents an enzyme from being made.The term “antisense molecule” as used herein encompasses any nucleicacid molecule that contains sequences that correspond to the codingstrand of an endogenous polypeptide. An antisense molecule also can haveflanking sequences (e.g., regulatory sequences). Thus antisensemolecules can be ribozymes or antisense oligonucleotides. A ribozyme canhave any general structure including, without limitation, hairpin,hammerhead, or axhead structures, provided the molecule cleaves RNA.

Yeast having a reduced enzymatic activity can be identified using manymethods. For example, yeast having reduced glycerol-3-phosphatedehydrogenase activity can be easily identified using common methods,which may include, for example, measuring glycerol formation via liquidchromatography.

Overexpression of Heterologous Genes

Methods for overexpressing a polypeptide from a native or heterologousnucleic acid molecule are well known. Such methods include, withoutlimitation, constructing a nucleic acid sequence such that a regulatoryelement promotes the expression of a nucleic acid sequence that encodesthe desired polypeptide. Typically, regulatory elements are DNAsequences that regulate the expression of other DNA sequences at thelevel of transcription. Thus, regulatory elements include, withoutlimitation, promoters, enhancers, and the like. For example, theexogenous genes can be under the control of an inducible promoter or aconstitutive promoter. Moreover, methods for expressing a polypeptidefrom an exogenous nucleic acid molecule in yeast are well known. Forexample, nucleic acid constructs that are used for the expression ofexogenous polypeptides within Kluyveromyces and Saccharomyces are wellknown (see, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529, forKluyveromyces and, e.g., Gellissen et al., Gene 190(1):87-97 (1997) forSaccharomyces). Yeast plasmids have a selectable marker and an origin ofreplication. In addition certain plasmids may also contain a centromericsequence. These centromeric plasmids are generally a single or low copyplasmid. Plasmids without a centromeric sequence and utilizing either a2 micron (S. cerevisiae) or 1.6 micron (K. lactis) replication originare high copy plasmids. The selectable marker can be eitherprototrophic, such as HIS3, TRP1, LEU2, URA3 or ADE2, or antibioticresistance, such as, bar, ble, hph, or kan.

In another embodiment, heterologous control elements can be used toactivate or repress expression of endogenous genes. Additionally, whenexpression is to be repressed or eliminated, the gene for the relevantenzyme, protein or RNA can be eliminated by known deletion techniques.

As described herein, any yeast within the scope of the disclosure can beidentified by selection techniques specific to the particular enzymebeing expressed, over-expressed or repressed. Methods of identifying thestrains with the desired phenotype are well known to those skilled inthe art. Such methods include, without limitation, PCR, RT-PCR, andnucleic acid hybridization techniques such as Northern and Southernanalysis, altered growth capabilities on a particular substrate or inthe presence of a particular substrate, a chemical compound, a selectionagent and the like. In some cases, immunohistochemistry and biochemicaltechniques can be used to determine if a cell contains a particularnucleic acid by detecting the expression of the encoded polypeptide. Forexample, an antibody having specificity for an encoded enzyme can beused to determine whether or not a particular yeast cell contains thatencoded enzyme. Further, biochemical techniques can be used to determineif a cell contains a particular nucleic acid molecule encoding anenzymatic polypeptide by detecting a product produced as a result of theexpression of the enzymatic polypeptide. For example, transforming acell with a vector encoding acetolactate synthase and detectingincreased acetolactate concentrations compared to a cell without thevector indicates that the vector is both present and that the geneproduct is active. Methods for detecting specific enzymatic activitiesor the presence of particular products are well known to those skilledin the art. For example, the presence of acetolactate can be determinedas described by Hugenholtz and Starrenburg, 1992, Appl. Micro. Biot.38:17-22.

Increase of Enzymatic Activity

Yeast microorganisms of the invention may be further engineered to haveincreased activity of enzymes. The term “increased” as used herein withrespect to a particular enzymatic activity refers to a higher level ofenzymatic activity than that measured in a comparable yeast cell of thesame species. For example, overexpression of a specific enzyme can leadto an increased level of activity in the cells for that enzyme.Increased activities for enzymes involved in glycolysis or theisobutanol pathway would result in increased productivity and yield ofisobutanol.

Methods to increase enzymatic activity are known to those skilled in theart. Such techniques may include increasing the expression of the enzymeby increased copy number and/or use of a strong promoter, introductionof mutations to relieve negative regulation of the enzyme, introductionof specific mutations to increase specific activity and/or decrease theKm for the substrate, or by directed evolution. See, e.g., Methods inMolecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press(2003).

Microorganism Characterized by Producing Isobutanol at High Yield

For a biocatalyst to produce isobutanol most economically, it is desiredto produce a high yield. Preferably, the only product produced isisobutanol. Extra products lead to a reduction in product yield and anincrease in capital and operating costs, particularly if the extraproducts have little or no value. Extra products also require additionalcapital and operating costs to separate these products from isobutanol.

The microorganism may convert one or more carbon sources derived frombiomass into isobutanol with a yield of greater than 5% of theoretical.In one embodiment, the yield is greater than 10%. In one embodiment, theyield is greater than 50% of theoretical. In one embodiment, the yieldis greater than 60% of theoretical. In another embodiment, the yield isgreater than 70% of theoretical. In yet another embodiment, the yield isgreater than 80% of theoretical. In yet another embodiment, the yield isgreater than 85% of theoretical. In yet another embodiment, the yield isgreater than 90% of theoretical. In yet another embodiment, the yield isgreater than 95% of theoretical. In still another embodiment, the yieldis greater than 97.5% of theoretical.

More specifically, the microorganism converts glucose, which can bederived from biomass into isobutanol with a yield of greater than 5% oftheoretical. In one embodiment, the yield is greater than 10% oftheoretical. In one embodiment, the yield is greater than 50% oftheoretical. In one embodiment the yield is greater than 60% oftheoretical. In another embodiment, the yield is greater than 70% oftheoretical. In yet another embodiment, the yield is greater than 80% oftheoretical. In yet another embodiment, the yield is greater than 85% oftheoretical. In yet another embodiment the yield is greater than 90% oftheoretical. In yet another embodiment, the yield is greater than 95% oftheoretical. In still another embodiment, the yield is greater than97.5% of theoretical

Microorganism Characterized by Production of Isobutanol from PyruvateVia an Overexpressed Isobutanol Pathway and a Pdc-Minus and Gpd-MinusPhenotype

In yeast, the conversion of pyruvate to acetaldehyde is a major drain onthe pyruvate pool, and, hence, a major source of competition with theisobutanol pathway. This reaction is catalyzed by the pyruvatedecarboxylase (PDC) enzyme. Reduction of this enzymatic activity in theyeast microorganism results in an increased availability of pyruvate andreducing equivalents to the isobutanol pathway and may improveisobutanol production and yield in a yeast microorganism that expressesa pyruvate-dependent isobutanol pathway.

Reduction of PDC activity can be accomplished by 1) mutation or deletionof a positive transcriptional regulator for the structural genesencoding for PDC or 2) mutation or deletion of all PDC genes in a givenorganism. The term “transcriptional regulator” can specify a protein ornucleic acid that works in trans to increase or to decrease thetranscription of a different locus in the genome. For example, in S.cerevisiae, the PDC2 gene, which encodes for a positive transcriptionalregulator of PDC1,5,6 genes can be deleted; a S. cerevisiae in which thePDC2 gene is deleted is reported to have only ˜10% of wildtype PDCactivity (Hohmann, 1993, Mol Gen Genet 241:657-66). Alternatively, forexample, all structural genes for PDC (e.g. in S. cerevisiae, PDC1,PDC5, and PDC6, or in K. lactis, PDC1) are deleted.

Crabtree-positive yeast strains such as S. cerevisiae strain thatcontains disruptions in all three of the PDC alleles no longer produceethanol by fermentation. However, a downstream product of the reactioncatalyzed by PDC, acetyl-CoA, is needed for anabolic production ofnecessary molecules. Therefore, the Pdc− mutant is unable to grow solelyon glucose, and requires a two-carbon carbon source, either ethanol oracetate, to synthesize acetyl-CoA (Flikweert et al., 1999, FEMSMicrobiol Lett. 174: 73-9; and van Maris et al., 2004, Appl EnvironMicrobiol. 70: 159-66).

Thus, in an embodiment, such a Crabtree-positive yeast strain may beevolved to generate variants of the PDC mutant yeast that do not havethe requirement for a two-carbon molecule and has a growth rate similarto wild type on glucose. Any method, including chemostat evolution orserial dilution may be utilized to generate variants of strains withdeletion of three PDC alleles that can grow on glucose as the solecarbon source at a rate similar to wild type (van Maris et al., 2004,Appl Envir Micro 70: 159-66).

Another byproduct that would decrease yield of isobutanol is glycerol.Glycerol is produced by 1) the reduction of the glycolysis intermediate,dihydroxyacetone phosphate (DHAP), to glycerol-3-phosphate (G3P) via theoxidation of NADH to NAD⁺ by Glycerol-3-phosphate dehydrogenase (GPD)followed by 2) the dephosphorylation of glycerol-3-phosphate to glycerolby glycerol-3-phosphatase (GPP). Production of glycerol results in lossof carbons as well as reducing equivalents. Reduction of GPD activitywould increase yield of isobutanol. Reduction of GPD activity inaddition to PDC activity would further increase yield of isobutanol.Reduction of glycerol production has been reported to increase yield ofethanol production (Nissen et al., 2000, Yeast 16, 463-74; Nevoigt etal., Method of modifying a yeast cell for the production of ethanol,WO/2009/056984). Disruption of this pathway has also been reported toincrease yield of lactate in a yeast engineered to produce lactateinstead of ethanol (Dundon et al., Yeast cells having disrupted pathwayfrom dihydroxyacetone phosphate to glycerol, US 2009/0053782).

In one embodiment, the microorganism is a Crabtree-positive yeast withreduced or no GPD activity. In another embodiment, the microorganism isa crabtree positive yeast with reduced or no GPD activity, and expressesan isobutanol biosynthetic pathway and produces isobutanol. In yetanother embodiment, the microorganism is a Crabtree-positive yeast withreduced or no GPD activity and with reduced or no PDC activity. Inanother embodiment, the microorganism is a crabtree positive yeast withreduced or no GPD activity, with reduced or no PDC activity, andexpresses an isobutanol biosynthetic pathway and produces isobutanol.

In another embodiment, the microorganism is a Crabtree-negative yeastwith reduced or no GPD activity. In another embodiment, themicroorganism is a Crabtree-negative yeast with reduced or no GPDactivity, expresses the isobutanol biosynthetic pathway and producesisobutanol. In yet another embodiment, the microorganism is aCrabtree-negative yeast with reduced or no GPD activity and with reducedor no PDC activity. In another embodiment, the microorganism is aCrabtree-negative yeast with reduced or no GPD activity, with reduced orno PDC activity, expresses an isobutanol biosynthetic pathway andproduces isobutanol.

PDC-minus/GPD-minus yeast production strains are described in co-pendingapplications U.S. Ser. No. 12/343,375 (published as US 2009/0226991),U.S. Ser. No. 12/696,645, and U.S. Ser. No. 12/820,505, which claimpriority to U.S. Provisional Application 61/016,483, all of which areherein incorporated by reference in their entireties for all purposes.

Method of Using Microorganism for High-Yield Isobutanol Fermentation

In a method to produce isobutanol from a carbon source at high yield,the yeast microorganism is cultured in an appropriate culture mediumcontaining a carbon source.

Another exemplary embodiment provides a method for producing isobutanolcomprising a recombinant yeast microorganism of the invention in asuitable culture medium containing a carbon source that can be convertedto isobutanol by the yeast microorganism of the invention.

In certain embodiments, the method further includes isolating isobutanolfrom the culture medium. For example, isobutanol may be isolated fromthe culture medium by any method known to those skilled in the art, suchas distillation, pervaporation, or liquid-liquid extraction, includingmethods disclosed in co-pending applications U.S. Ser. No. 12/342,992(published as US 2009/0171129) and PCT/US08/88187 (published asWO/2009/086391), which are herein incorporated by reference in theirentireties for all purposes.

This invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication, as well as the Figure and the Sequence Listing, areincorporated herein by reference for all purposes.

EXAMPLES General Methods

TABLE 1 Amino acid sequences disclosed herein. SEQ ID NO Protein,Accession No. 1 E. coli IlvC, NP_418222 2 S. cerevisiae Ilv5, NP_0134593 Oryza sativa KARI, NP_001056384 4 Methanococcus maripaludis KARI,YP_001097443 5 Acidiphilium cryptum KARI, YP_001235669 6 Chlamydomonasreinhardtii KARI, XP_001702649 7 Picrophilus torridus KARI, YP_023851 8Zymomonas mobilis KARI, YP_162876 9 c-myc epitope tag 10 Thermotogapetrophila RKU-1 dihydroxyacid dehydratase (DHAD), YP_001243973.1 11Victivallis vadensis ATCC BAA-548 dihydroxyacid dehydratase (DHAD),ZP_01924101.1 12 Termite group 1 bacterium phylotype Rs-D17dihydroxyacid dehydratase (DHAD), YP_001956631.1 13 Yarrowia lipolyticadihydroxyacid dehydratase (DHAD), XP_502180.2 14 Francisella tularensissubsp. tularensis WY96-3418 dihydroxyacid dehydratase (DHAD),YP_001122023.1 15 Arabidopsis thaliana dihydroxyacid dehydratase (DHAD),AAK64025.1 16 Candidatus Koribacter versatilis Ellin345 dihydroxyaciddehydratase (DHAD), YP_592184.1 (Acidobacter) 17 Gramella forsetiiKT0803 dihydroxyacid dehydratase (DHAD), YP_862145.1 18 Lactococcuslactis subsp. lactis Il1403 dihydroxyacid dehydratase (DHAD),NP_267379.1 19 Saccharopolyspora erythraea NRRL 2338 dihydroxyaciddehydratase (DHAD), YP_001103528.2 20 Saccharomyces cerevisiae Ilv3,NP_012550.1 21 Piromyces sp E2 ilvD 22 Ralstonia eutropha JMP134 ilvD,YP_298150.1 23 Chromohalobacter salexigens ilvD, YP_573197.1 24Picrophilus torridus DSM9790 ilvD, YP_024215.1 25 Sulfolobus tokodaiistr. 7 dihydroxyacid dehydratase (DHAD), NP_378168.1 26 Saccharomycescerevisiae Ilv3ΔN 27 P(I/L)XXXGX(I/L)XIL (conserved motif described inExample 17) 28 PIKXXGX(I/L)XIL (conserved motif described in Example 17)

TABLE 2 Nucleic acid sequences disclosed herein. SEQ ID NO Gene,Accession No. 87 Lactococcus lactis subsp. lactis Il1403 (Ll_ilvD) 88Saccharomyces cerevisiae ILV3 (ScILV3(FL)) 89 Saccharomyces cerevisiaeILV3ΔN (ScILV3ΔN) 90 Gramella forsetii KT0803 (Gf_ilvD) 91Saccharopolyspora erythraea NRRL 2338 (Se_ilvD) 92 Candidatus Koribacterversatilis Ellin345 ilvD (Acidobacter) 93 Piromcyes sp E2 ilvD(Piromyces ilvD) 94 Ralstonia eutropha JMP134 ilvD, (Re_ilvD) 95Chromohalobacter salexigens ilvD, (Cs_ilvD) 96 Picrophilus torridusDSM9790 ilvD, (Pt_ilvD) 97 Sulfolobus tokodail str. 7 ilvD, (St_ilvD) 98E. coli ilvC^(Q110V), (Ec_ilvC(Q110V)) 99 Lactococcus lactis kivD,(Ll_kivD) 100 S. cerevisiae ILV5, (ScILV5)

Determination of Optical Density.

The optical density of the yeast cultures is determined at 600 nm usinga DU 800 spectrophotometer (Beckman-Coulter, Fullerton, Calif., USA).Samples are diluted as necessary to yield an optical density of between0.1 and 0.8.

Gas Chromatography.

Analysis of volatile organic compounds, including ethanol and isobutanolwas performed on a HP 5890 gas chromatograph fitted with an HP 7673Autosampler, a DB-FFAP column (J&W; 30 m length, 0.32 mm ID, 0.25_μMfilm thickness) or equivalent connected to a flame ionization detector(FID). The temperature program was as follows: 200° C. for the injector,300° C. for the detector, 50° C. oven for 1 minute, 31° C./minutegradient to 140° C., and then hold for 2.5 min. Analysis was performedusing authentic standards (>99%, obtained from Sigma-Aldrich), and a5-point calibration curve with 1-pentanol as the internal standard.

High Performance Liquid Chromatography for Quantitative Analysis ofGlucose and Organic Acids.

Analysis of glucose and organic acids was performed on a HP-1100 HighPerformance Liquid Chromatography system equipped with an Aminex HPX-87HIon Exclusion column (Bio-Rad, 300×7.8 mm) or equivalent and an H⁺cation guard column (Bio-Rad) or equivalent. Organic acids were detectedusing an HP-1100 UV detector (210 nm, 8 nm 360 nm reference) whileglucose was detected using an HP-1100 refractive index detector. Thecolumn temperature was 60° C. This method was Isocratic with 0.008 Nsulfuric acid in water as the mobile phase. Flow was set at 1 mL/min.Injection volume was 20 μL and the run time was 30 minutes.

High Performance Liquid Chromatography for Quantitative Analysis ofKetoisovalerate and Isobutyraldehyde.

Analysis of the DNPH derivatives of ketoisovalerate and isobutyraldehydewas performed on a HP-1100 High Performance Liquid Chromatography systemequipped with a Hewlett Packard 1200 HPLC stack column (Agilent EclipseXDB-18, 150×4.0 mm; 5 μm particles [P/N #993967-902] and C18 Guardcartridge). The analytes were detected using an HP-1100 UV detector at360 nm The column temperature was 50° C. This method was isocratic with0.1% H₃PO₄ and 70% acetonitrile in water as mobile phase. Flow was setat 3 mL/min. Injection size was 10 μL and the run time was 2 minutes.

Molecular Biology and Bacterial Cell Culture.

Standard molecular biology methods for cloning and plasmid constructionare generally used, unless otherwise noted (Sambrook, J., Russel, D. W.Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press).

Standard recombinant DNA and molecular biology techniques used in theExamples are well known in the art and are described by Sambrook, J.,Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; and by T. J.Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984) andby Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub.by Greene Publishing Assoc. and Wiley-Interscience (1987).

General materials and methods suitable for the routine maintenance andgrowth of bacterial cultures are well known in the art. Techniquessuitable for use in the following examples may be found as set out inManual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E.Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R.Krieg and G. Briggs Phillips, eds.), American Society for Microbiology,Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, Second Edition, Sinauer Associates,Inc., Sunderland, Mass. (1989).

Yeast transformations—S. cerevisiae.

S. cerevisiae strains were transformed by the Lithium Acetate method(Gietz et al., Nucleic Acids Res. 27:69-74 (1992): Cells from 50 mL YPDcultures (YPGaI for valine auxotrophs) were collected by centrifugation(2700 rcf, 2 minutes, 25° C.) once the cultures reached an OD₆₀₀ of 1.0.The cells were washed cells with 50 mL sterile water and collected bycentrifugation at 2700 rcf for 2 minutes at 25° C. The cells were washedagain with 25 mL sterile water and collected cells by centrifugation at2700 rcf for 2 minutes at 25° C. The cells were resuspended in 1 mL of100 mM lithium acetate and transferred to a 1.5 mL eppendorf tube. Thecells were collected cells by centrifugation for 20 sec at 18,000 rcf,25° C. The cells were resuspended cells in a volume of 100 mM lithiumacetate that was approximately 4× the volume of the cell pellet. Amixture of DNA (final volume of 15 μl with sterile water), 72 μl 50%PEG, 10 μl 1 M lithium acetate, and 3 μl denatured salmon sperm DNA wasprepared for each transformation. In a 1.5 mL tube, 15 μl of the cellsuspension was added to the DNA mixture (85 μl), and the transformationsuspension was vortexed with 5 short pulses. The transformation wasincubated at 30 minutes at 30° C., followed by incubation for 22 minutesat 42° C. The cells were collected by centrifugation for 20 sec at18,000 rcf, 25° C. The cells were resuspended in 100 μl SOS (1 Msorbitol, 34% (v/v) YP (1% yeast extract, 2% peptone), 6.5 mM CaCl₂) or100 μl YP (1% yeast extract, 2% peptone) and spread over an appropriateselective plate.

Yeast Transformations—K. lactis.

K. lactis cells were transformed according to a slightly modifiedversion of the protocol as described by Kooistra et al., Yeast 21:781-792 (2004). Saturated overnight-grown cultures of K. lactis cellswere diluted 1:50 into 100 mL YPD and were placed in 30° C. shaker (250rpm) and grown for 4-5 hours until the culture reached an OD₆₀₀ of0.3-0.5. Cells were collected by centrifugation (2 min, 3000×g) andwashed with 50 ml cold sterile EB (electroporation buffer; 10 mMTris-HCl, pH 7.5, 270 mM sucrose, 1 mM MgCl₂) at 4° C. Cells wereresuspended in 50 mL YPD that contained 25 mM DTT and 20 mM HEPES, pH8.0 Cells were transferred back into flasks used to grow cells andincubated in 30° C. incubator (without shaking) for 30 minutes. Cellswere then collected by centrifugation (2 minutes, 3000×g) and washedwith 10 mL ice-cold sterile EB, as above. Cells were then resuspendedusing one cell pellet volume of ice-cold sterile EB. Sixty microlitersof cells were mixed with plasmid DNA and incubated on ice for 15minutes. For targeted integrations, or transformation of linear DNA,approximately 200-400 ng of non-specific, short (50-500 bp) linear DNAfragments were added to 300-400 ng of the linearized integrating DNAconstruct. This DNA was either provided by gel-purified Alul-digestedsalmon sperm DNA, or a mixture of annealed primers 35+36 (yielding a ˜85bp linear duplex fragment). Cells were transferred cells to a chilledelectroporation (2 mm) cuvette and pulsed using a BioRad Gene Pulser at1 kV, 400Ω, and 25 uF. The cell suspension was immediately transferredto a 14 mL round-bottom Falcon tube with 1 mL room temperature YPD andallowed to incubate vertically at 30° C., 225 RPM for at 6-18 h. Cellswere collected in an 1.7 mL by centrifugation for 10 seconds at maximumspeed, and resuspended with 150 μL YPD before being spread ontoappropriate selection plates.

Yeast Colony PCR with FailSafe™ PCR System(EPICENTRE® Biotechnologies,

Madison, Wis.; Catalog #FS99250): Cells from each colony were added to20 μl of colony PCR mix (per reaction mix contains 6.8 μl water, 1.5 μlof each primer, 0.2 μl of FailSafe PCR Enzyme Mix and 10 μl 2× FailSafeMaster Mix). Unless otherwise noted, 2× FailSafe Master Mix E was used.The PCR reactions were incubated in a thermocycler using the followingtouchdown PCR conditions: 1 cycle of 94° C.×2 min, 10 cycles of 94°C.×20 s, 63°−54° C.×20 s (decrease 1° C. per cycle), 72° C.×60 s, 40cycles of 94° C.×20 s, 53° C.×20 s, 72° C.×60 s and 1 cycle of 72° C.×5min.

Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, Calif.; Catalog#D4002) Protocol:

DNA fragments were recovered from agarose gels according tomanufacturer's protocol.

Zymo Research DNA Clean and Concentrator Kit (Zymo Research, Orange,Calif.; Catalog #D4004) Protocol:

DNA fragments were purified according to manufacturer's protocol.

Preparation of Cell Lysates for In Vitro Enzyme Assays.

To grow cultures for cell lysates, triplicate independent cultures ofthe desired strain were grown overnight in 3 mL of the appropriatemedium at 30° C., 250 rpm. The following day, the overnight cultureswere diluted into 50 mL fresh medium in 250 mL baffle-bottomedErlenmeyer flasks and incubated at 30° C. at 250 rpm. Cells were grownfor at least 4 generations and the cultures were harvested in mid logphase (OD₆₀₀ of 1-3) The cells of each culture were collected bycentrifugation (2700×g, 5 min, 4° C.). The cell pellets were washed byresuspending in 20 mL of ice cold water. The cells were centrifuged at2700×g, 4° C. for 5 min. All supernatant was removed from each tube andthe tubes were frozen at −80° C. until use.

Lysates were prepared by thawing each cell pellet on ice and preparing a20% (w/v) cell suspension in lysis buffer. The lysis buffer was variedfor each enzyme assay and consisted of: 0.1 M Tris-HCl pH 8.0, 5 mMMgSO₄, for DHAD assays, 50 mM potassium phosphate buffer pH 6.0, 1 mMMgSO₄ for ALS assays, 250 mM KPO₄ pH 7.5, 10 mM MgCl₂ for KARI assays,50 mM NaHPO₄, 5 mM MgCl₂, for KIVD assays. 10 μL of Yeast/FungalProtease Arrest solution (G Biosciences, catalog #788-333) per 1 mL oflysis buffer was used. 800 microliters of cell suspension were added to1 mL of 0.5 mm glass beads that had been placed in a chilled 1.5 mLtube. Cells were lysed by bead beating (6 rounds, 1 minute per round, 30beats per second) with 2 minutes chilling on ice in between rounds. Thetubes were then centrifuged (20,000×g, 15 min) to pellet debris and thesupernatants (cell lysates) were retained in fresh tubes on ice. Theprotein concentration of each lysate was measured using the BioRadBradford protein assay reagent (BioRad, Hercules, Calif.) according tomanufacturer's instructions.

Preparation of Fractionated Lysates from S. cerevisiae Strains for InVitro Enzyme Assays.

To grow cultures for cell fractionated cell lysates, triplicateindependent cultures of the desired strain were grown overnight in 3 mLof the appropriate medium at 30° C., 250 rpm. The following day, theovernight cultures were used to inoculate 1 L cultures of each strainwhich were grown in the appropriate medium at 30° C. at 250 rpm untilthey reached an OD₆₀₀ of approximately 2. The cells were collected bycentrifugation (1600×g, 2 min) and the culture medium was decanted. Thecell pellets were resuspended in 50 mL sterile deionized water,collected by centrifugation (1600×g, 2 min), and the supernatant wasdiscarded.

To obtain spheroplasts, the cell pellets were resuspended in 0.1 MTris-SO₄, pH 9.3, to a final concentration of 0.1 g/mL, and DTT wasadded to a final concentration of 10 mM. Cells were incubated withgentle (60 rev/min) agitation on an orbital shaker for 20 min at 30° C.,and the cells were then collect by centrifugation (1600×g, 2 min) andthe supernatant discarded. Each cell pellet was resuspended inspheroplasting buffer, which consists of (final concentrations): 1.2Msorbitol (Amresco, catalog #0691), 20 mM potassium phosphate pH 7.4) andthen collected by centrifugation (1600×g, 10 min). Each cell pellet wasresuspended in spheroplasting buffer to a final concentration of 0.1 gcells/mL in a 500 mL centrifuge bottle, and 50 mg of Zymolyase 20T(Seikagaku Biobusiness, Code#120491) was added to each cell suspension.The suspensions were incubated overnight (approximately 16 hrs) at 30°C. with gentle agitation (60 rev/min) on an orbital shaker. The efficacyof spheroplasting was ascertained by diluting an aliquot of each cellsuspension 1:10 in either sterile water or in spheroplasting buffer, andcomparing the aliquots microscopically (under 40× magnification). In allcases, >90% of the water-diluted cells lysed, indicating efficientspheroplasting. The spheroplasts were centrifuged (3000×g, 10 min, 20°C.), and the supernatant was discarded. Each cell pellet was resuspendedin 50 mL spheroplast buffer without Zymolyase, and cells were collectedby centrifugation (3000×g, 10 min, 20° C.).

To fractionate spheroplasts, the cells were resuspended to a finalconcentration of 0.5 g/mL in ice cold mitochondrial isolation buffer(MIB), consisting of (final concentration): 0.6M D-mannitol (BD DifcoCat#217020), 20 mM HEPES-KOH, pH 7.4. For each 1 mL of resulting cellsuspension, 0.01 mL of Yeast/Fungal Protease Arrest solution (GBiosciences, catalog #788-333) was added. The cell suspension wassubjected to 35 strokes of a Dounce homogenizer with the B (tight)pestle, and the resulting cell suspension was centrifuged (2500×g, 10min, 4° C.) to collect cell debris and unbroken cells and spheroplasts.Following centrifugation, 2 mL of each sample (1 mL of the pGV1900transformed cells) were saved in a 2 mL centrifuge tube on ice anddesignated the “W” (for Whole cell extract) fraction, while theremaining supernatant was transferred to a clean, ice-cold 35 mLOakridge screw-cap tube and centrifuged (12,000×g, 20 min, 4° C.) topellet mitochondria and other organellar structures. Followingcentrifugation, 5 mL of each resulting supernatant was transferred to aclean tube on ice, being careful to avoid the small, loose pellet, andlabelled the “S” (soluble cytosol) fraction. The resulting pellets wereresuspended in MIB containing Protease Arrest solution, and werelabelled the “P” (“pellet”) fractions. The BioRad Protein Assay reagent(BioRad, Hercules, Calif.) was used according to manufacturer'sinstructions to determine the protein concentration of each fraction.

Preparation of Fractionated Lysates from K. lactis Strains for In VitroEnzyme Assays

Cultures (20 mL YPD) were inoculated with yeast cells (GEVO1742 andGEVO1829) and incubated at 30° C. while shaking at 250 RPM until theyreached late-log to stationary phase (OD₆₀₀ of approximately 10). Cellsfrom the 20 mL cultures were used to inoculate a 250 mL YPD culture atan OD₆₀₀ of approximately 0.2. The cultures were incubated at 30° C.while shaking at 250 RPM until they reached mid-log (OD₆₀₀ ˜2).

To prepare spheroplasts, the cells were collected in 500 mL bottles at5000×g for 5 minutes at room temperature. The pellets were resuspendedwith 8 mL Spheroplasting Buffer A (25 mM potassium phosphate (pH 7.5), 1mM MgCl₂, 1 mM EDTA, 1.25 mM TPP, 1 mM DTT) without sorbitol andtransferred to pre-weighed 50 mL tubes. The cells were collected at1600×g for 5 minutes at room temperature. The cells were resuspendedwith 8 mL of Spheroplasting Buffer A with 2.5 M Sorbitol (AmrescoCode#0691) and protease inhibitor (G Biosciences Yeast/FungalProteaseArrest™ (Catalog #788-333)). Approximately 5 mg of Zymolyase 20TZymolyase 20T (Seikagaku Biobusiness Code#120491) was added to each cellsuspension. The suspensions were incubated at 30° C. with gentleagitation (e.g. 50 RPM), with the tube on its side for good mixing, for1-2 hours. The efficiency of formation of spheroplasts was verified bydilution of the spheroplast suspension 1:10 into Spheroplasting Buffer Awith 2.5 M sorbitol and 1:10 in water. Spheroplasts should remain intactwhen diluted into the buffer but appear fuzzy or completely disappearwhen diluted into water. The spheroplasts were collected at 1600×g for 7minutes at 4° C. The spheroplasts were gently washed with 2 mL ofSpheroplasting Buffer A with 2.5 M sorbitol and protease inhibitor, andcollected at 1600×g for 7 minutes at 4° C. The spheroplasts wereresuspended in 2 mL of Spheroplasting Buffer A with 2.5 M sorbitol andprotease inhibitor.

To fractionate the spheroplasts, 8 mL of Spheroplasting Buffer A with0.2 M sorbitol and protease inhibitor was slowly added to the cellsuspension, bringing the final concentration of Sorbitol to 0.66 M. Thespheroplasts were broken with 10 strokes using a B (tight fitting)pestle in a 15 mL Dounce homogenizer (Bellco Glass, Inc. Cat#1984-10015)on ice. The homogenate was transferred to a 50 mL tube, and the celldebris was collected by centrifugation at 4° C. for 10 minutes at1600×g. The supernatant was transferred to a 15 mL tube with a pipette.This supernatant is the “W” fraction. 5 mL of this “W” fraction wastransferred to a 35 mL Oakridge tube and centrifuged at 48,000×g for 20minutes at 4° C. The resulting supernatant was transferred to a 15 mLtube and labeled “S.” The pellet was resuspended in 5 mL ofSpheroplasting Buffer A with 0.66 M Sorbitol and protease inhibitor andlabeled “P.” All fractions were stored on ice at 4° C. while in use. TheBioRad Protein Assay reagant (BioRad, Hercules, Calif.) was usedaccording to manufacturer's instructions to determine the proteinconcentration of each fraction.

ALS Assay.

Cell lysates were prepared and protein concentrations were determined asdescribed above. The colorimetric ALS Assay (FAD-independent) performedhere was based on the assay described in Hugenholtz, J. and Starrenburg,J. C., Appl. Microbiol. Biotechnol. (1992) 38:17-22. Reaction buffer wasprepared by mixing 900 μl 1M potassium phosphate buffer pH 6.0, 180 μl100 mM MgSO₄, 180 μl 100 mM TPP, 3.96 ml 500 mM pyruvate and 12.78 mlwater. For the no substrate control, the volume of pyruvate was replacedwith water. Lysates were prepared at a final protein concentration of 2μg/μl in Spheroplasting Buffer A with 0.66 M sorbitol. To 900 μL ALSBuffer, 100 μL of lysate was added and incubated at 30° C. for 30 min.Acetoin standards were also prepared at concentrations of 2 mM, 1 mM,0.5 mM, and 0 mM. From each sample and standard, 175 μL was transferredto a fresh 1.5 mL tube. To each sample and standard was added 25 μL 35%(v/v) H₂SO₄, and all were incubated at 37° C. for 30 mins. After theincubation, the following were added in order, to each standard andsample, with the solutions being mixed by vortexing in between eachaddition: 50 μL 50% (w/v) NaOH, 50 μL 0.5% creatine, and 50 μL 5%1-naphthol (in 2.5N NaOH). The samples and standards were incubated atroom temperature for 1 hour, being mixed by vortexing every 15 minutes.To a 96 well, half-area, UV-Star, transparent, flat-bottom plate(Catalog #675801, Greiner Bio One, Frickenhausen, Germany), 100 uL ofeach sample or standard was transferred, and the samples were analyzedby a plate reader by measuring absorbance at 530 nm.

KARI Assay.

Cell lysates were prepared and protein concentrations were determined asdescribed above. Acetolactate substrate was made by mixing 50 μl ofethyl-2 acetoxy-2-methyl-acetoacetate with 990 μl of water. Then 10 μlof 2 N NaOH was sequentially added, with vortex mixing betweenadditions, until 260 μl of NaOH was added. The acetolactate was agitatedat room temperature for 20 min and then held on ice. NADPH was preparedin 0.01N NaOH (to improve stability) to a concentration of 50 mM. Theconcentration was determined by reading the OD of a diluted sample at340 nm in a spectrophotometer and using the molar extinction coefficientof 6.22 M⁻¹ cm⁻¹ to calculate the actual concentration (the OD₃₄₀ of a100 μM solution of NAD(P)H should be 0.622). Three buffers were preparedand held on ice. Reaction buffer contained 250 mM KPO₄ pH 7.5, 10 mMMgCl₂, 1 mM DTT, 10 mM acetolactate, and 0.2 mM NADPH. No substratebuffer contained everything except the acetolactate. No NAD(P)H buffercontained everything except the NADPH. Reactions were performed intriplicate using 10 μl of cell extract with 90 μl of reaction buffer ina 96-well plate in a SpectraMax 340PC multi-plate reader (MolecularDevices, Sunnyvale, Calif.). The reaction was followed at 340 nm bymeasuring a kinetic curve for 5 minutes, with OD readings taken every 10seconds. The reactions were performed at 30° C. The reactions wereperformed in complete, no substrate, and no NAD(P)H buffers. The V_(max)for each extract was determined after subtracting the background readingof the no substrate control from the reading in complete buffer.

DHAD Assay.

Cell lysates were prepared and protein concentrations were determined asdescribed above. The DHAD activity of each lysate was ascertained asfollows. In a fresh 1.5 mL centrifuge tube, 50 μL of each lysate wasmixed with 50 μL of 0.1 M 2,3-dihydroxyisovalerate (DHIV), 25 μL of 0.1M MgSO₄, and 375 μL of 0.05M Tris-HCl pH 8.0, and the mixture wasincubated for 30 min at 35° C. Each tube was then heated to 95° C. for 5min to inactivate any enzymatic activity, and the solution wascentrifuged (16,000×g for 5 min) to pellet insoluble debris. To preparesamples for analysis, 100 μL of each reaction were mixed with 100 μL ofa solution consisting of 4 parts 15 mM dinitrophenyl hydrazine (DNPH) inacetonitrile with 1 part 50 mM citric acid, pH 3.0, and the mixture washeated to 70° C. for 30 min in a thermocycler. The solution was thenanalyzed by HPLC as described above in General Methods to quantitate theconcentration of ketoisovalerate (KIV) present in the sample.

KIVD Assay.

Cell lysates were prepared and protein concentrations were determined asdescribed above. KIVD Assay buffer, containing 1 Roche ProteaseInhibitor tablet per 5 mL buffer, was added to each cell pellet tocreate a 20% (w/v) cell suspension. The KIVD assay buffer was preparedat a final concentration of 0.05 M NaHPO₄*H₂O, 5 mM MgCl₂*8H₂O, and 1.5mM Thiamin pyrophosphate chloride. The reaction substrate,α-keto-isovalerate (3-methyl-2-oxobutanoic acid, Acros Organics), wasadded where appropriate at 30 mM. Lysates were diluted in reactionbuffer at a final protein concentration of 0.1 μg/μL. To 1.5 mL tubes,50 μL of lysate (5 μg of protein) was mixed with 200 μL of reactionbuffer with or without substrate. The reactions were incubated at 37° C.for 20 minutes, and the reactions were immediately filtered through a 2μm filter plate. The filtered samples were diluted 1:10 in water, and100 μL of the 1:10 dilution was mixed with 100 μL of derivatizationreagent in a 0.2 ml thin-wall PCR tubes. Derivatization reagent wasprepared by mixing 4 ml of 2,4-Dinitrophenyl Hydrazine (DNPH) in 15 mMin HPLC-grade Acetonitrile with 1 ml 50 mM Citric Acid Buffer, pH 3. Thesamples were incubated at 70° C. for 30 minutes. The samples wereanalyzed by HPLC.

ADH Assay.

Cell lysates were prepared and protein concentrations were determined asdescribed above. Assays (set up in triplicate for each lysate) contained10 μL of each lysate (or an appropriate dilution of each lysate) plus 90μL of reaction buffer, which consisted of (final concentrations presentin 1× reaction buffer): 0.1M Tris-HCl pH 7.5, 10 mM MgC₁₂, 1 mM DTT, 0.2mM NADH (or NADPH, where indicated; each diluted from a 4.4 mMspectrophotometrically-confirmed stock), and 11 mM isobutyraldehyde.Where indicated, as controls a parallel set of assay reactions were setup using reaction buffer lacking isobutyraldehyde and/or NAD(P)H, asindicated. For experiments measuring the acetaldehyde-dependentoxidation of NAD(P)H, reaction buffer was prepared in which acetaldehydewas substituted for isobutyraldehyde. In these cases, the reactionbuffer contained at least 11 mM acetaldehyde, although the exact amountpresent is an estimate due to the inherent difficulties of pipettingacetaldehyde solution. Finally, in some cases a parallel set ofreactions lacking yeast cell lysate was included as a negative control.After being added (using a multi-channel pipet) to the wells of a96-well plate, the reactions were immediately placed into a plate readerthat had been pre-warmed to 30° C., and the absorbance at 340 nm wasmeasured every 12 seconds over a period of 300 seconds. Kineticparameters were computed from assays with linear slopes (wherenecessary, assays were repeated with appropriate dilutions to obtainlinear NAD(P)H consumption curves).

Composition of Culture Media

Drugs: When indicated, G418 (Calbiochem, Gibbstown, N.J.) was added at0.2 g/L, Phleomycin (InvivoGen, San Diego, Calif.) was added at 7.5mg/L, Hygromycin (InvivoGen, San Diego, Calif.) was added at 0.2 g/L,and 5-fluoro-orotic acid (FOA; Toronto Research Chemicals, North York,Ontario, Canada) was added at 1 g/L.

YP: 1% (w/v) yeast extract, 2% (w/v) peptone.

YPD: YP containing 2% (w/v) glucose unless otherwise noted,

YPGal: YP containing 2% (w/v) galactose

YPE: YP containing 2% (w/v) Ethanol.

SC media: 6.7 g/L Difco™ Yeast Nitrogen Base, 14 g/L Sigma™ SyntheticDropout Media supplement (includes amino acids and nutrients excludinghistidine, tryptophan, uracil, and leucine; Sigma-Aldrich, St. Louis,Mo.), 0.076 g/L histidine, 0.076 g/L tryptophan, 0.380 g/L leucine, and0.076 g/L uracil. Drop-out versions of SC media is made by omitting oneor more of histidine (H), tryptophan (W), leucine (L), or uracil (U orUra). When indicated, SC media are supplemented with additionalisoleucine (9xI; 0.684 g/L), valine (9xV; 0.684 g/L) or both isoleucineand valine (9xIV). SCD is SC containing 2% (w/v) glucose unlessotherwise noted, SCGal is SC containing 2% (w/v) galactose and SCE is SCcontaining 2% (w/v) ethanol. For example, SCD-Ura+9xIV would be composedof 6.7 g/L Difco™ Yeast Nitrogen Base, 14 g/L Sigma™ Synthetic DropoutMedia supplement (includes amino acids and nutrients excludinghistidine, tryptophan, uracil, and leucine), 0.076 g/L histidine, 0.076g/L tryptophan, 0.380 g/L leucine, 0.684 g/L isoleucine, 0.684 g/Lvaline, and 20 g/L glucose.

SCD-V+9xI: 6.7 g/L Difco™ Yeast Nitrogen Base, 0.076 g/L Adeninehemisulfate, 0.076 g/L Alanine 0.076 g/L, Arginine hydrochloride, 0.076g/L Asparagine monohydrate, 0.076 g/L Aspartic acid, 0.076 g/L Cysteinehydrochloride monohydrate, 0.076 g/L Glutamic acid monosodium salt,0.076 g/L Glutamine, 0.076 g/L Glycine, 0.076 g/L myo-lnositol, 0.76 g/LIsoleucine, 0.076 g/L Lysine monohydrochloride, 0.076 g/L Methionine,0.008 g/L p-Aminobenzoic acid potassium salt, 0.076 g/L Phenylalanine,0.076 g/L Proline, 0.076 g/L Serine, 0.076 g/L Threonine, 0.076 g/LTyrosine disodium salt, and 20 g/L glucose.

YNB: 6.7 g/L Difco™ Yeast Nitrogen Base supplemented with indicatednutrients as follows: histidine (H; 0.076 g/L), tryptophan (W; 0.076g/L), leucine (L; 0.380 g/L), uracil (U or Ura; 0.076 g/L), isoleucine(1; 0.076 g/L), valine (V; 0.076 g/L), and casamino acids (CAA; 10 g/L).When indicated, YNB media are supplemented with higher amounts ofisoleucine (10xI=0.76 g/L), valine (10xV=0.76 g/L) or both isoleucineand valine (10xIV). YNBD is YNB containing 2% (w/v) glucose unlessotherwise noted, YNBGal is YNB containing 2% (w/v) galactose and YNBE isYNB containing 2% (w/v) ethanol. For example, YNBGal+HWLU+10xI+G418would be composed of 6.7 g/L Difco™ Yeast Nitrogen Base, 0.076 g/Lhistidine, 0.076 g/L tryptophan, 0.380 g/L leucine, 0.076 g/L uracil,0.76 g/L isoleucine, 0.2 g/L G418, and 20 g/L galactose.

Plates: Solid versions of the above described media contain 2% (w/v)agar.

Example 1 Isobutanol Pathway is Partially Cytosolic when Expressed inYeast

The purpose of this example is to illustrate that three enzymes in theisobutanol biosynthetic pathway (acetolactate synthase, ketoisovaleratedecarboxylase, and isobutanol dehydrogenase) are localized to thecytosol when expressed in yeast.

TABLE 3 Genotype of strains disclosed in Example 1. GEVO No.Genotype/Source 1287 K. lactis ATCC 200826 MAT α uraA1 trp1 leu2 lysA1ade1 lac4-8 [pKD1] 1742 K. lactis ATCC 200826 MAT α uraA1 trp1 leu2lysA1 ade1 lac4-8 [pKD1] pdc1::Kan^(R) 1829 K. lactis ATCC 200826 MAT αuraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1] pdc1::kan^(R){P_(TDH3):Ec_ilvC- ΔN; P_(TEF1):Ec_ilvD-ΔN(codon optimized for K.lactis):ScLEU2 integrated} {P_(TEF1):Ll_kivD; P_(TDH3)ScADH7:KmURA3integrated} {P_(CUP1-1): Bs_alsS, TRP1 random integrated}

TABLE 4 Plasmids disclosed in Example 1. pGV No. Genotype pGV1503ScTEF1promoter-kanR bla, pUC ori (GEVO) pGV1537 KlPDC1 promoter region +Klpdc1 3′UTR sequence, ScTEF1promoter-kanR bla, pUC ori (GEVO) pGV1590TEF1 promoter:Ll-kivd (codon optimized for E. coli):TDH3promoter:ADH7:CYC1 terminator, Km-URA3, 1.6 micron ori, bla, pUC ori(GEVO) pGV1726 CUP1 promoter:Bs-alsS:CYC1 terminator, TRP1, bla, pUC-oripGV1727 TEF1 promoter:Ec-ilvDΔN (codon optimized for K. lactis):TDH3promoter:Ec-ilvCΔN:CYC1 terminator, LEU2, bla, pUC ori (GEVO)

Plasmids

pGV1503 contains an S. cerevisiae TEF1 promoter region driving aG418-resistance gene (kan^(R)).

pGV1537 was constructed by inserting an (AatII plus MfeI)-digested PCRproduct containing approximately 500 bp each of KIPDC1 5′ and 3′untranslated regions, into (AatII plus EcoRI)-digested pGV1503. Theinsert was generated by SOE-PCR. First, the KIPDC1 5′ and 3′untranslated regions were amplified from K. lactis genomic DNA by primerpairs 1006+1016 and 1017+1009, respectively. Primers 1016 and 1017 weredesigned to have overlapping sequences. The two fragments were thenjoined by PCR using primers 1006+1009.

pGV1590 is a K. lactis plasmid for expression of the L. lactis kivD andthe S. cerevisiae ADH7. Expression of the L. lactis kivD is driven bythe S. cerevisiae TEF1 promoter and expression of the S. cerevisiae ADH7is driven by the S. cerevisiae TDH3 promoter. pGV1590 was generated bycloning a SalI-NotI fragment carrying the S. cerevisiae ADH7 gene intothe XhoI-NotI sites of pGV1585. The S. cerevisiae ADH7 gene fragmentoriginated as a PCR product from S. cerevisiae genomic DNA using primers410 and 411.

pGV1726 is a yeast integration plasmid (utilizing the S. cerevisiae TRP1gene as selection marker) for random integration (i.e. for K. lactis).This plasmid does not carry a yeast replication origin, thus is unableto replicate episomally. This plasmid also carries the B. subtilis alsSgene, whose expression is under the control of the S. cerevisiae CUP1promoter. pGV1726 was generated by cloning a SacI-NgoMIV fragmentcarrying the S. cerevisiae CUP1 promoter, Bs-alsS ORF and the CYC1terminator into the same sites of pGV1645. The vector, pGV1645, is a K.lactis expression plasmid that was used for expression of the B.subtilis alsS under the control of the K. lactis PDC1 promoter. Thisplasmid also carries the S. cerevisiae TRP1 gene as a selection markerand the 1.6 micron replication origin. Digestion of pGV1645 with SacIand NgoMIV removes the K. lactis PDC1 promoter, B. subtilis alsS, CYC1terminator and the 1.6 micron origin of replication. The insert fragmentcarrying the S. cerevisiae CUP1 promoter, B. subtilis alsS ORF and theCYC1 terminator was obtained from pGV1649 via digestion with SacI andNgoMIV. The CUP1 promoter originated as a PCR product from S. cerevisiaegenomic DNA using primers 637 and 638. The B. subtilis alsS originatedas a PCR product from B. subtilis genomic DNA using primers 767 and 697.

pGV1727 is a yeast integration plasmid (utilizing the S. cerevisiae LEU2gene as selection marker) for random integration (i.e. for K. lactis).This plasmid does not carry a yeast replication origin, thus is unableto replicate episomally. This plasmid carries the E. coli ilvDΔN andilvCΔN genes, whose expressions are under the control of the S.cerevisiae TEF1 and TDH3 promoters respectively. The E. coli ilvDΔN is ashortened version of E. coli ilvD where the sequence coding for thefirst 24 amino acids, which encodes for a putative mitochondrialtargeting sequence, was removed. Likewise, the E. coli ilvCΔN is ashortened version of E. coli ilvC where the sequence coding for thefirst 22 amino acids, which is predicted to function as a mitochondrialtargeting sequence was removed. pGV1727 was generated by cloning aXhoI-NgoMIV fragment carrying the E. coli ilvCΔN gene and the CYC1terminator into the same sites of pGV1635. The vector, pGV1635, is a K.lactis expression plasmid that was used for expression of the E. coliilvDΔN gene under the control of the S. cerevisiae TEF1 promoter. TheilvDΔN gene is followed by the TDH3 promoter, a short MCS (includes anXhoI site), the CYC1 terminator and the 1.6 micron replication origin.This plasmid carries the S. cerevisiae LEU2 gene as a selection marker.Digestion of pGV1635 with XhoI and NgoMIV removes the CYC1 terminatorand the 1.6 micron replication origin. This sequence was replaced by theinsert fragment carrying the E. coli ilvCΔN and the CYC1 terminatorwhich was obtained from pGV1677 digested with XhoI and NgoMIV. The E.coli ilvDΔN originated as a PCR product from pGV1578 (plasmid carryingE. coli ilvD codon optimized for K. lactis from DNA2.0, Menlo Park,Calif.) using primers 1151 and 1152. The E. coli ilvCΔN originated as aPCR product from pGV1160 (plasmid carrying the full length E. coli ilvCgene) using primers 1149 and 1150. The E. coli ilvC in pGV1160originated as a PCR product from E. coli genomic DNA using primers 387and 388.

GEVO1287 was transformed with PmlI-digested pGV1537, yielding GEVO1742.GEVO1829 was constructed by sequentially transforming GEVO1742 with genefragments from pGV1590, pGV1727, and pGV1726 following the standardlithium acetate protocol. First, a 7.8 kb fragment of pGV1590 generatedby digestion with NgoMIV and MfeI was transformed into GEVO1742. Next,this transformant strain was transformed with pGV1727 (FIG. 4) that hadbeen linearized by digestion with BcgI. Finally, this transformantstrains was transformed with pGV1726 that had been linearized bydigestion with AhdI. The final transformant was GEVO1829.

Cellular fractions were prepared from GEVO1742 and GEVO1829 as describedabove. The protein concentration used to calculate specific activitiesfrom all three fractions (“W,” “S,” and “P”) was measured for the “W”fraction. Below are the results for the assays measuring isobutanoldehydrogenase, acetolactate synthase, and ketoisovalerate decarboxylaseactivities.

Alcohol Dehydrogenase (ADH) Assay

The results from the assay are summarized in Table 5. The “W” fractionand the “S” fraction of the pathway carrying strain (GEVO1829) containedat least three times the NADPH dependent alcohol dehydrogenase activityfound in the same fractions of GEVO1742. The “W” and “S” fractions ofGEVO1829 contained more than four times the activity present in the “P”fraction. These data indicated that S. cerevisiae Adh7 activity waspredominantly localized to the cytosol.

TABLE 5 Alcohol Dehydrogenase Activity. Specific Alcohol SampleDehydrogenase Activity (U/mg protein) 1742 W 0.08 ± 0.00 1742 S 0.07 ±0.02 1742 P  0.03 ± 0.012 1829 W 0.26 ± 0.00 1829 S 0.25 ± 0.02 1829 P0.04 ± 0.02

Acetolactate Synthase (ALS) Assay

The results from the assay are summarized in Table 6. The “W” and “S”fractions of the isobutanol pathway carrying strain (GEVO1829) containedALS activity, while no activity was detected in the same fractions ofGEVO1742. The “W” and “S” fractions contained three times higher ALSactivity than the “P” fraction. These data indicated that B. subtilisALS activity was predominantly localized to the cytosol.

TABLE 6 Acetolactate Synthase Activity. Sample Specific AcetolactateSynthase Activity (U/mg protein) 1742 W 0.00 ± 0.00 1742 S 0.00 ± 0.001742 P 0.00 ± 0.00 1829 W 0.10 ± 0.01 1829 S 0.10 ± 0.00 1829 P 0.03 ±0.00

Ketoisovalerate Decarboxylase (KIVD) Assay

The results from the assay are summarized in Table 7. The “W” and “S”fractions of the isobutanol pathway carrying strain (GEVO1829) contained8-10 times greater activity than in the same fractions of GEVO1742.Furthermore, the activity in “S” fraction was 45× higher than what wasdetected in “P” fraction. These data indicated that L. lactis KIVDactivity was predominantly localized in the cytosol.

TABLE 7 Ketoisovalerate decarboxylase (KIVD) Assay. Sample SpecificKetoisovalerate Decarboxylase Activity (U/mg protein) 1742 W 0.05 ± 0.001742 S 0.05 ± 0.04 1742 P 0.03 ± 0.00 1829 W 0.38 ± 0.02 1829 S 0.45 ±0.04 1829 P 0.01 ± 0.00

Example 2 Construction of an ILV3 Deletion Mutant

The purpose of this example is to describe the construction of an ILV3deletion mutant of S. cerevisiae, GEVO2244.

TABLE 8 Genotype of strains disclosed in Example 2. GEVO No.Genotype/Source GEVO1147 K. lactis, NRRL Y-1140, (obtained from USDA)GEVO1188 S. cerevisiae, CEN.PK, (obtained from Euroscarf); MATα ura3leu2 his3 trp1 GEVO2145 S. cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1ilv3::Kl_URA3 GEVO2244 S. cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1ilv3Δ

TABLE 9 Plasmids disclosed in Example 2. Plasmid name Genotype pUC19bla, pUC-ori (obtained from Invitrogen) pGV1299 K. lactis URA3, bla,pUC-ori (GEVO)

Plasmid pGV1299 was constructed by cloning the K. lactis URA3 gene intopUC19. The K. lactis URA3 was obtained by PCR using primers 575 and 576from K. lactis genomic DNA. The PCR product was digested with EcoRI andBamHI and cloned into pUC19 which was similarly digested. The K. lactisURA3 insert was sequenced (Laragen Inc) to confirm correct sequence.

The ilv3::KI_URA3 integration cassette contained, from 5′ to 3′, thefollowing: 1) a 80 bp homology to ILV3 (position +158 to 237) thatfunctions as the 5′ targeting sequence for the integration, 2) the K.lactis URA3 marker gene, 3) a 60 bp homology to a region ILV3 (position−21 to +39) that is further upstream of the 5′ targeting sequence tofacilitate loop-out of the K. lactis URA3 marker, and 4) a 221 bphomology to the 3′ region of ILV3 (position +1759 to 1979) thatfunctions as the 3′ targeting sequence for the integration. Thiscassette was generated by SOE-PCR. The K. lactis URA3 gene was amplifiedfrom pGV1299 using primers 1887 and 1888. Only the 3′ region of ILV3 wasinitially amplified using primers 1623 and 1892 from genomic DNA andthis product was used as template to amplify the 3′ region of ILV3 usingprimers 1889 and 1890. The K. lactis URA3 and the 3′ region of ILV3 werecombined by SOE-PCR using primers 1886 and 1890.

GEVO1188 was transformed with the ilv3::KI_URA3 cassette described aboveand plated onto YNBD+W+CAA (−Ura) plates. Initially, eight colonies(#1-8) were patched onto YNBD+HUWLIV plates and then replica plated ontoYNBD+HUWLI (−V) plates to test for valine auxotrophy. As none of theseexhibited valine auxotrophy, an additional eight colonies (#9-16) werestreaked out for single colonies and 3 or 4 isolates (A through C or D)from each streak were tested for valine auxotrophy. Isolates A-C fromclone #12 exhibited valine auxotrophy.

These isolates were tested for the correct integrations by colony PCRusing primer pairs 1916+1920 and 1917+1921 for the 5′ and 3′ junctions,respectively. Correct sized bands were observed with clones #12A throughC with primer pair 1916+1920. Correct sized bands were observed withclone 12A when FailSafe Master Mix A or C was used with primer pair1917+1921. Clone #12A was designated as GEVO2145. The valineauxotrophies of GEVO2145 were reconfirmed by streaking them ontoSCD+9xIV and SCD-V+9xI plates. GEVO2145 exhibited no growth on themedium lacking valine (SCD-V+9xI) while it grew on medium supplementedwith valine (SCD+9xIV). The parent strain, GEVO1188, grew on both media.

GEVO2145 was streaked onto YNBE+W+CAA+FOA to isolate strains in whichthe K. lactis URA3 had been excised through homologous recombination,i.e. “looped out”. Five FOA resistant clones (A-E) were tested forauxotrophies for valine and uracil. All five clones exhibitedauxotrophies to both nutrients. Clone A was designated GEVO2244. ColonyPCR using primers 1891 and 1892 with FailSafe Buffer C was performed andthe loss of the KI_URA3 cassette was confirmed.

Example 3 DHAD Activity is Localized to Mitochondria

The purpose of this Example is to demonstrate that the DHAD activityencoded by ScILV3 is localized to the mitochondria.

TABLE 9 Genotype of strains disclosed in Example 3. GEVO No.Genotype/Source Gevo2244 S. cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1ilv3Δ

TABLE 10 Plasmids disclosed in Example 3. pGV No. Genotype pGV1106 pUCori, bla (AmpR), 2micron ori, URA3, TDH3 promoter- Myctag-polylinker-CYC1 terminator pGV1900 pUC ori, bla (AmpR), 2micron ori,URA3, TEF1 promoter-ScILV3(FL)

Plasmid pGV1106 is a variant of p426GPD (described in Mumberg et al,1995, Gene 119-122). To obtain pGV1106, annealed oligos 271 and 272 wereligated into p426GPD that had been digested with SpeI and XhoI, and theinserted DNA was confirmed by sequencing.

Plasmid pGV1900 was generated by amplifying the full-length, nativeScILV3 nucleotide sequence from S. cerevisiae strain CEN.PK genomic DNAusing primers 1617 and 1618. The resulting 1.76 kb fragment, whichcontained the complete ScILV3 coding sequence (SEQ ID NO: 88) flanked by5′ SalI and 3′ BamHI restriction site sequences was digested with SalIand BamHI and ligated into pGV1662 (described in Example 6) which hadbeen digested with SalI and BamHI.

To measure DHAD activities present in fractionated cell extracts,GEVO2244 was transformed singly with either pGV1106, which served as anempty vector control, or with pGV1900, which is an expression plasmidfor ScILV3.

An independent clonal transformant of each plasmid was isolated, and a 1L culture of each strain was grown in SCGaI-Ura+9xIV at 30° C. at 250rpm. The OD₆₀₀ was noted, the cells were collected by centrifugation(1600×g, 2 min) and the culture medium was decanted. The cell pelletswere resuspended in 50 mL sterile deionized water, collected bycentrifugation (1600×g, 2 min), and the supernatant was discarded. TheOD₆₀₀ and total wet cell pellet weight of each culture are listed inTable 11, below:

TABLE 11 OD₆₀₀ and pellet mass (g) of strain GEVO2244 transformed withthe indicated plasmids. Pellet mass Plasmid OD₆₀₀ (g) pGV1106 2.2 7.6pGV1900 1.3 3.8

To obtain spheroplasts, the cell pellets were resuspended in 0.1MTris-SO₄, pH 9.3, to a final concentration of 0.1 g/mL, and DTT wasadded to a final concentration of 10 mM. Cells were incubated withgentle (60 rev/min) agitation on an orbital shaker for 20 min at 30° C.,and the cells were then collect by centrifugation (1600×g, 2 min) andthe supernatant discarded. Each cell pellet was resuspended inspheroplasting buffer, which consists of (final concentrations): 1.2Msorbitol (Amresco, catalog #0691), 20 mM potassium phosphate pH 7.4) andthen collected by centrifugation (1600×g, 10 min). Each cell pellet wasresuspended in spheroplasting buffer to a final concentration of 0.1 gcells/mL in a 500 mL centrifuge bottle, and 50 mg of Zymolyase 20T(Seikagaku Biobusiness, Code#120491) was added to each cell suspension.The suspensions were incubated overnight (˜16 hrs) at 30° C. with gentleagitation (60 rev/min) on an orbital shaker. The efficacy ofspheroplasting was ascertained by diluting an aliquot of each cellsuspension 1:10 in either sterile water or in spheroplasting buffer, andcomparing the aliquots microscopically (under 40× magnification). In allcases, >90% of the water-diluted cells lysed, indicating efficientspheroplasting. The spheroplasts were centrifuged (3000×g, 10 min, 20°C.), and the supernatant was discarded. Each cell pellet was resuspendedin 50 mL spheroplast buffer without Zymolyase, and cells were collectedby centrifugation (3000×g, 10 min, 20° C.).

To fractionate spheroplasts, the cells were resuspended to a finalconcentration of 0.5 g/mL in ice cold mitochondrial isolation buffer(MIB), consisting of (final concentration): 0.6M D-mannitol (BD DifcoCat#217020), 20 mM HEPES-KOH, pH 7.4. For each 1 mL of resulting cellsuspension, 0.01 mL of Yeast/Fungal Protease Arrest solution (GBiosciences, catalog #788-333) was added. The cell suspension wassubjected to 35 strokes of a Dounce homogenizer with the B (tight)pestle, and the resulting cell suspension was centrifuged (2500 g, 10min, 4° C.) to collect cell debris and unbroken cells and spheroplasts.Following centrifugation, 2 mL of each sample (1 mL of the pGV1900transformed cells) were saved in a 2 mL centrifuge tube on ice anddesignated the “W” (for Whole cell extract) fraction, while theremaining supernatant was transferred to a clean, ice-cold 35 mLOakridge screw-cap tube and centrifuged (12,000×g, 20 min, 4° C.) topellet mitochondria and other organellar structures. Followingcentrifugation, 5 mL of each resulting supernatant was transferred to aclean tube on ice, being careful to avoid the small, loose pellet, andlabelled the “S” (soluble cytosol) fraction. The resulting pellets wereresuspended in MIB containing Protease Arrest solution, and werelabelled the “P” (“pellet”) fractions. Protein from the “P” fraction wasreleased after dilution 1:5 in DHAD assay buffer (see above) by rapidmixing in a 1.5 mL tube with a Retsch Ball Mill MM301 in the presence of0.1 mM glass beads. The mixing was performed 4 times for 1 minute.

The BioRad Protein Assay reagant (BioRad, Hercules, Calif.) was usedaccording to manufacturer's instructions to determine the proteinconcentration of each fraction.

The DHAD activity of each fraction was ascertained as described in themethods above.

TABLE 12 Specific activities (KIV generation) and ratios of specificactivities from fractionated lysates of S. cerevisiae strain GEVO2244carrying plasmids to overexpress the indicated DHAD homolog. Each datapoint is the result of triplicate samples. Sp. Activity Lysate (pGV#[U/mg protein and fraction*) DHAD in fraction] Std. Dev. 1106 W — n.d.1106 S — n.d. 1106 P — n.d. 1900 W ScILV3(FL) 0.0096 0.0018 1900 SScILV3(FL) 0.0052 0.0004 1900 P ScILV3(FL) 0.0340 0.0029

Cells overexpressing the full-length, native S. cerevisiae Ilv3contained in a greater proportion of the specific DHAD activity in themitochondrial fraction (P) versus the cytosolic fraction (S).

Example 4 Replacing Current Mitochondrially Targeted Isobutanol PathwayEnzymes with Fungal Homologs or Functional Analogs that are Targeted tothe Cytosol

The purpose of this example is to illustrate that fungal homologs ofisobutanol a pathway enzymes exhibit cytosolic activity.

TABLE 13 Genotype of strains disclosed in Example 4. GEVO No.Genotype/Source 1187 MATa ura3-52 leu2-3_112 his3Δ1 trp1-289 ADE2CEN.PK2-1C 2280 MATa ura3-52 leu2-3_112 his3Δ1 trp1-289 ADE2 CEN.PK2-1Cintegrated pGV1730 at PDC1 locus 2618 MATa ura3-52 leu2-3_112 his3Δ1trp1-289 ADE2 CEN.PK2-1C integrated pGV2114 at PDC1 locus 2621 MATaura3-52 leu2-3_112 his3Δ1 trp1-289 ADE2 CEN.PK2-1C integrated pGV2117 atPDC1 locus 2622 MATa ura3-52 leu2-3_112 his3Δ1 trp1-289 ADE2 CEN.PK2-1Cintegrated pGV2118 at PDC1 locus

TABLE 14 Plasmids disclosed in Example 4. pGV No. Genotype 1730P_(Cup1-1)1:Bs_alsS, pUC ORI, Amp^(R), TRP1, PDC1 3′-fragment-NruI-PDC15′-fragment. 2114 P_(Cup1-1)1:Bs_alsScoSc, pUC ORI, Amp^(R), TRP1, PDC13′-fragment-NruI-PDC1 5′-fragment. 2117 P_(Cup1-1)1:Ta_alsS, pUC ORI,Amp^(R), TRP1, PDC1 3′-fragment-NruI-PDC1 5′-fragment. 2118P_(Cup1-1)1:Ts_alsS, pUC ORI, Amp^(R), TRP1, PDC1 3′-fragment-NruI-PDC15′-fragment.

Yeast AHASs are normally mitochondrial, thus favoring fungal ALS enzymesfor as cytosolically functional isobutanol pathway enzymes. Sequenceanalysis by Le and Choi (Bull. Korean Chem. Soc. (2005) 26:916-920)showed that there is a conserved sequence ‘RFDDR’ found in AHASs that isnot conserved among ALSs. This sequence is likely involved inFAD-binding by AHASs and thus could be used to distinguish between theFAD-dependent AHASs and the FAD-independent ALSs. Using this region todistinguish between AHASs and ALSs BLAST searches of fungal sequencedatabases were performed and resulted in the identification of ALShomologs from several fungal species (Magnaporthe grisea, Phaeosphaerianodorum, Trichoderma atroviride (SEQ ID NO: 71), Talaromyces stipitatus(SEQ ID NO: 72), Penicillium marneffei, and Glomerella graminicola). Ofthese sequences, the ALS homologs from M. grisea, P. nodorum, T.atroviride and T. stipitatus are predicted to be cytoplasmic by MitoprotII v.1.101 as described in the paper M. G. Claros, P. Vincens.Computational method to predict mitochondrially imported proteins andtheir targeting sequences. Eur. J. Biochem. 241, 779-786 (1996).

Fungal ALS genes were synthesized by DNA 2.0 with codon optimizationbiased for S. cerevisiae. The following ALS constructs were made andtested for ALS activity by assaying acetoin in the media during a growthtimecourse. All ALS genes were cloned into the integration vectorpGV1730 (SEQ ID NO: 69) as described herein.

Plasmid pGV1730 is a yeast integration plasmid used to replace the PDC1gene in S. cerevisiae with the B. subtilis alsS gene (SEQ ID NO: 70)(not codon optimized for S. cerevisiae) expressed using the S.cerevisiae CUP1 promoter. This plasmid carries the S. cerevisiae TRP1gene as a selection marker.

Construction of pGV2114: pGV1730 was treated with BamHI and SalI and the4932 bp vector fragment was purified by gel electrophoresis asdescribed. The B. subtilis AlsS (codon-optimized for expression in S.cerevisiae) gene was ligated to the pGV1730 vector fragment as a BamHIand SalI 1722 bp fragment using standard methods with an approximately5:1 insert:vector molar ratio and transformed into TOP10 chemicallycompetent E. coli cells. Plasmid DNA was isolated and correct cloneswere confirmed using restriction enzyme analysis.

Construction of pGV2117. pGV1730 was treated with BamHI and SalI and the4932 bp vector fragment was purified by gel electrophoresis asdescribed. The T. atroviride ALS gene was ligated to the pGV1730 vectorfragment as a BamHI and SalI 1686 bp fragment using standard methodswith an approximately 5:1 insert:vector molar ratio and transformed intoTOP10 chemically competent E. coli cells. Plasmid DNA was isolated andcorrect clones were confirmed using restriction enzyme analysis.

Construction of pGV2118. pGV1730 was treated with BamHI and SalI and the4932 bp vector fragment was purified by gel electrophoresis asdescribed. The T. stipitatus ALS gene was ligated to the pGV1730 vectorfragment as a BamHI and SalI 1707 bp fragment using standard methodswith an approximately 5:1 insert:vector molar ratio and transformed intoTOP10 chemically competent E. coli cells. Plasmid DNA was isolated andcorrect clones were confirmed using restriction enzyme analysis.

All yeast strains were constructed by treating the plasmid to beintegrated with NruI and then transforming the plasmid according to thestandard yeast transformation protocol as described herein.Transformants were selected by plating transformed cells onto SCD-Wmedia and growing at 30° C. for 2 days. Primary transformants weresingle colony purified and then tested for correct integration usingcolony PCR. Colony PCR was performed using the Yeast colony PCR to checkfor proper integration of the integrative plasmids used the FailSafe™PCR System (EPICENTRE® Biotechnologies, Madison, Wis.; Catalog #FS99250)according to the manufacturer protocol The PCR reactions were incubatedin a thermocycler using the following conditions: 1 cycle of 94° C. for2 min, 40 cycles of 94° C. for 30 s, 53° C. for 30 s, 72° C. for 60 sand 1 cycle of 72° C. for 10 min. Presence of the positive PCR productwas assessed using agarose gel electrophoresis. Primer pairs for the5′-end and 3′-end integration sites contained one primer on the plasmidand one primer in the genome.

Yeast strains GEVO1187, 2280, 2618, 2621 and 2622 were grown in YPDovernight at 30° C. A 100 mL culture was inoculated to 1 OD/mL and splitinto 2 50 mL cultures. This was the time zero. One of the 50 mL culturesreceived 500 μM CuSO₄ at time 2 hours and the other did not. Timepointsconsisted of removing 1 mL at times 0, 2, 2.5, 3, 4, 7.5, and 23 hours.At each timepoint the OD₆₀₀ was determined and acetoin concentrationswere determined using GC as described in the General Methods. Before GCsamples were treated with H₂SO₄ to convert intermediates to acetoin. Thegraph shows the acetoin concentrations in the media of the strains inwhich transcription of the ALS genes was induced by CuSO₄. The acetoinvalues were normalized to cell OD. Both the T. stipitatus ALS and the T.atroviride ALS showed increased levels of acetoin as compared to the noALS control (FIG. 2).

ALS activity in whole cell lysates is determined as described in GeneralMethods. Activity in mitochondrial/organellar (P) and cytosolic (S)fractions and whole cell (W) lysates is assayed as described in GeneralMethods

Example 5 Replacing Current Mitochondrially Targeted Isobutanol PathwayEnzymes with Homologs or Functional Analogs from Anaerobic Fungi

The purpose of this example is to illustrate that homologues ofisobutanol a pathway enzymes from anaerobic fungi exhibit cytosolicactivity.

TABLE 15 Genotype of strains disclosed in Example 5. GEVO No. GenotypeGEVO2244 S. cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1 ilv3Δ

TABLE 16 Plasmids disclosed in Example 5. Plasmid name Genotype pGV1106pUC ori, bla (AmpR), 2 μm ori, URA3, TDH3 promoter-Myctag-polylinker-CYC1 terminator pGV1662 pUC ori, bla (AmpR), 2 μm ori,URA3, TEF1 promoter-(kivD) pGV1855 pUC ori, bla (AmpR), 2 μm ori, URA3,TEF1 promoter-Ll_ilvD

Plasmid pGV1106 is described in Example 3, above.

Plasmid pGV1662 (SEQ ID NO: 81) served as the parental plasmid ofpGV1855, pGV1900, and pGV2019. The salient features of pGV1662 includethe yeast 2 micron origin of replication, the URA3 selectable marker,and the ScTEF1 promoter sequence followed by restriction sites intowhich an ORF can be cloned to permit its expression under the regulationof the TEF1 promoter.

Plasmid pGV1855 contains the L. lactis ilvD. The L. lactis ilvD sequencewas synthesized (DNA2.0, Menlo Park, Calif.) and included a unique SalIand a NotI site at the 5′ and 3′ end of the coding sequence,respectively. The synthesized DNA was digested with SalI and NotI andligated into vector pGV1662 that had been digested with SalI plus NotI,yielding pGV1855.

The DHAD homolog (ilvD) from the anaerobic fungi Piromyces sp. E2 has apredicted MTS of 49 amino acids at the N-terminus. Thus, a nucleotidesequence encoding the Piromyces ilvD lacking the N-terminal 49 aminoacids and with a start codon placed at the N-terminus was synthesized(SEQ ID NO: 73). In addition, a SalI site and a BamHI site wereintroduced at the 5′ and 3′ ends of this ORF. This fragment was clonedinto the SalI and BamHI sites of pGV1662. The resulting plasmid wastransformed in to GEVO2242. An empty vector, pGV1106, is used as anegative control. Plasmid, pGV1855, expressing L. lactis ilvD is used asa positive control.

An independent clonal transformant of each plasmid is isolated, and a 1L culture of each strain is grown in SCGaI-Ura+9xIV at 30° C. at 250rpm. The OD₆₀₀ is noted, the cells are collected by centrifugation(1600×g, 2 min) and the culture medium is decanted. The cell pellets areresuspended in 50 mL sterile deionized water, collected bycentrifugation (1600×g, 2 min), and the supernatant is discarded.

To obtain spheroplasts, the cell pellets are resuspended in 0.1MTris-SO₄, pH 9.3, to a final concentration of 0.1 g/mL, and DTT is addedto a final concentration of 10 mM. Cells are incubated with gentle (60rev/min) agitation on an orbital shaker for 20 min at 30° C., and thecells are then collected by centrifugation (1600×g, 2 min) and thesupernatant discarded. Each cell pellet is resuspended in spheroplastingbuffer, which consists of (final concentrations): 1.2M sorbitol(Amresco, catalog #0691), 20 mM potassium phosphate pH 7.4) and thencollected by centrifugation (1600×g, 10 min). Each cell pellet isresuspended in spheroplasting buffer to a final concentration of 0.1 gcells/mL in a 500 mL centrifuge bottle and 50 mg of Zymolyase 20T(Seikagaku Biobusiness, Code#120491) is added to each cell suspension.The suspensions are incubated overnight (approximately 16 hrs) at 30° C.with gentle agitation (60 rev/min) on an orbital shaker. The efficacy ofspheroplasting is ascertained by diluting an aliquot of each cellsuspension 1:10 in either sterile water or in spheroplasting buffer, andcomparing the aliquots microscopically (under 40× magnification). Thespheroplasts are centrifuged (3000×g, 10 min, 20° C.), and thesupernatant is discarded. Each cell pellet is resuspended in 50 mLspheroplast buffer without Zymolyase and cells are collected bycentrifugation (3000×g, 10 min, 20° C.).

To fractionate spheroplasts, the cells are resuspended to a finalconcentration of 0.5 g/mL in ice cold mitochondrial isolation buffer(MIB), consisting of (final concentration): 0.6M D-mannitol (BD DifcoCat#217020), 20 mM HEPES-KOH, pH 7.4. For each 1 mL of resulting cellsuspension, 0.01 mL of Yeast/Fungal Protease Arrest solution (GBiosciences, catalog #788-333) is added. The cell suspension issubjected to 35 strokes of a Dounce homogenizer with the B (tight)pestle, and the resulting cell suspension is centrifuged (2500×g, 10min, 4° C.) to collect cell debris and unbroken cells and spheroplasts.Following centrifugation, 2 mL of each sample (1 mL of the pGV1900transformed cells) are saved in a 2 mL centrifuge tube on ice anddesignated the “W” (for Whole cell extract) fraction, while theremaining supernatant is transferred to a clean, ice-cold 35 mL Oakridgescrew-cap tube and centrifuged (12,000×g, 20 min, 4° C.) to pelletmitochondria and other organellar structures. Following centrifugation,5 mL of each resulting supernatant is transferred to a clean tube onice, being careful to avoid the small, loose pellet, and labelled the“S” (soluble cytosol) fraction. The resulting pellets are resuspended inMIB containing Protease Arrest solution, and are labelled the “P”(“pellet”) fractions. The protein concentration of each fraction isdetermined using the BioRad Protein Assay reagant (BioRad, Hercules,Calif.) according to manufacturer's instructions.

The DHAD activity of each fraction is ascertained using the DHAD assaysas described above in the General Methods.

Example 6 Modification of the N-Terminal Mitochondrial TargetingSequence of an Isobutanol Pathway Enzyme

The purpose of this example is to illustrate that removal ormodification of N-terminal mitochondrial targeting sequences allows forcytosolic activity of isobutanol pathway enzymes.

TABLE 17 Genotype of strains disclosed in Example 6. GEVO Genotype No.1803 MATa/alpha ura3/ura3 leu2/leu2 his3/his3 trp1/trp1 pdc1::Bs-alsS,TRP1/PDC1

TABLE 18 Plasmids disclosed in Example 6. Plasmid name RelevantGenes/Usage Genotype pGV1354 Plasmid that contains P_(TDH3):ILVΔN47:CYC1the Ilv5ΔN47 gene. term, bla, ColE1 ORI, URA3, 2 μ ori. pGV1662 Parentvector that has pTEF1::L. lactis Ampicillin resistance, kivD::CYC1 the 2μ origin, a URA3 gene, the term, bla, ColE1 ORI, TEF1 promoter, CYC1URA3, 2 μ ori. terminator region and an E. coli origin. It also has theL. lactis KivD gene that is removed by cutting the plasmid with SalI andNotI, and then gel purifying the vector portion. SalI and NotI were usedfor cloning genes to be expressed from the TEF1 promoter. pGV1810Plasmid that contains the pTEF1::ILV5::CYC1 full length ILV5 gene. Thiswas term, bla, used as a PCR template to ColE1 ORI, URA3, 2 μ ori.generate the Δ46-ilv5 mutant. pGV1831 Plasmid that contains pTEF1::ScIlv5 the Ilv5ΔN47 gene N47::CYC1 under control of the TEF1 term, bla,ColE1 ORI, promoter. URA3, 2 μ ori. pGV1833 Plasmid that containspTEF1::Sc ILV5:CYC1 the full length ILV5 gene under term, bla, controlof the TEF1 promoter. ColE1 ORI, URA3, 2 μ ori pGV1901 The S. cerevisiaeKARI pTEF1::Δ46ilv5 with the N-terminal KARI::CYC1 46 amino acid deleted(Δ46) cloned term, bla, ColE1 into pGV1662 at the SalI-NotI ORI, URA3, 2μ ori sites of the vector. The S. cerevisiae Δ46 KARI was a SalI-NotIfragment that was PCR amplified from pGV1810 using primers 1809 and1615. pGV1824 The E. coli coSc KARI pTEF1::E. coli coSc cloned intopGV1662 KARI:CYC1 term, bla, at SalI-BamHI sites of the vector. ColE1ORI, URA3, 2 μ ori

The yeast enzymes acetohydroxyacid synthase (AHAS; ILV2+ILV6),ketol-acid reductoisomerase (KARI; ILV5), and dihydroxyacid dehydratase(DHAD; ILV3) that carry out the first three steps of isobutanolproduction are physiologically localized to the mitochondria.Mitochondrial matrix proteins are typically targeted to the mitochondriaby an N-terminal mitochondrial targeting sequence (MTS), which is thencleaved off in the mitochondria resulting in the ‘mature’ form of theenzyme. N-terminal deletions of ILV5 have been shown to re-localize thisenzyme to the cytosol (Omura, 2008, Appl. Microbiol. Biotechnol. 78:503-513; Omura, WO/2009/078108 A1, hereby incorporated by reference inits entirety).

N-terminal mitochondria targeting sequences (MTS) are predicted byMitoProt II software; Claros et al., 1996, Eur. J. Biochem. 241:779-786. Two N-terminal deletions of the ILV5 gene was constructed, onemissing the first 46 amino acids and one missing the first 47 aminoacids.

pGV1831 was constructed as follows. pGV1662 was digested with SalI andNotI and the large fragment (6.3 Kb vector backbone) was gel purified byagarose gel electrophoresis. The Ilv5ΔN47 gene was excised from plasmidpGV1354 (SEQ ID NO: 80) using SalI and NotI. The ilv5ΔN47 gene fragment(1.06 Kb) was purified away from the larger vector fragment by agarosegel electrophoresis. The pGV1662 vector and ilv5ΔN47 insert were ligatedusing standard methods in an approximately 5:1 insert:vector molar ratioand transformed into TOP10 chemically competent E. coli cells. PlasmidDNA was isolated and correct clones were confirmed using restrictionenzyme analysis, namely generation of the correct insert size bydigesting clones with SalI and NotI enzymes. The clones were verified bysequencing with the primers 351, 1625, and 1626. Purified plasmid DNAwas transformed into S. cerevisiae strain GEVO1803 using a standardyeast transformation protocol.

pGV1833 was constructed as follows. pGV1662 was digested with SalI andNotI and the large fragment (6.3 Kb vector backbone) was gel purified byagarose gel electrophoresis. Primers 1615 and 1616 were used to amplifythe S. cerevisiae ILV5 gene from the plasmid template pGV1810 by PCR.The correct fragment size was verified with DNA gel electrophoresis (1.2Kb). The PCR product was purified after PCR using the Qiagen QIAquickPCR Purification Kit. The PCR product was then digested with XhoI andNotI to generate ends compatible with the pGV1662 backbone (the XhoI endof the PCR product is compatible with the SalI end of the vector,although the ligated DNA fragment can't be recut with either enzyme).After digestion, the PCR product was purified with a Qiagen QIAquick PCRPurification Kit. The two fragments were ligated using standard methodsin an approximately 5:1 insert:vector molar ratio and transformed intoTOP10 chemically competent E. coli cells. Plasmid DNA was isolated andcorrect clones were confirmed using restriction enzyme analysis. In thiscase, SacI plus NotI digestion yielded a fragment of the predicted size(1.6 Kb). The clones were verified by sequencing with the primers 351,1625, and 1626. Purified plasmid DNA was transformed into S. cerevisiaestrain GEVO1803.

pGV1901 was constructed as follows. pGV1662 was digested with SalI andNotI and the large fragment (6.3 Kb vector backbone) was gel purified byagarose gel electrophoresis. The ILV5 gene was amplified from pGV1810(SEQ ID NO: 82) using primers 1809 (which removes the first 46 aminoacids from the N-terminus while adding a methionine codon) and 1615. ThePCR product was digested with SalI and NotI. After digestion, the PCRproduct was purified on an agarose gel and the proper fragment (1.07 Kb)was recovered using the Zymoclean Gel DNA Recovery Kit. The pGV1662vector and Ilv5-Δ46 PCR products were ligated using standard methods inan approximately 5:1 insert:vector molar ratio and transformed intoTOP10 chemically competent E. coli cells. Plasmid DNA was isolated andcorrect clones were confirmed with PCR screening of colonies usingprimers 351 and 1577. The predicted correct PCR product was 580 bp. Theclones were sequenced using primers 351, 1625, and 1626. Purifiedplasmid DNA was transformed into S. cerevisiae strain GEVO1803 using thestandard yeast transformation protocol.

pGV1824 contains the E. coli ilvC gene that is codon optimized for S.cerevisiae cloned into the SalI and BamHI of pGV1662 as described above.The sequence of the codon optimized E. coli ilvC is found as SEQ ID NO:83.

Plasmids were transformed into the yeast strain GEVO1803 and anindividual colony was purified from each transformation. KARI assays ofwhole cell lysates were performed at pH 7.5 as described in GeneralMethods. Results are shown in FIG. 3.

KARI activity in mitochondrial/organellar (P) and cytosolic (S)fractions and whole cell (W) lysates is assayed as described in GeneralMethods

Example 7 Scaffolding Two or More Isobutanol Pathway Enzymes

The purpose of this example is to illustrate how isobutanol pathwayenzymes can be scaffolded in order to localize them to the cytosol.

Cellulolytic microorganisms utilize a scaffolded enzyme complex called acellulosome. In such a complex, numerous enzymes are docked to a singlescaffold protein, called a scaffoldin, which contain multiple bindingdomains called cohesin domains. Each cohesin domain interacts with adockerin domain. In a cellulosome complex, each cellulytic enzyme alsohas a dockerin domain that allows it to bind to the scaffoldin.

The cohesin domains of a scaffoldin protein, for example, CipA fromClostridium thermocellum, can be expressed in yeast. The dockerindomains from the cellulolytic enzymes from the same organism, forexample Xyn10B, can be fused to the isobutanol enzymes and the fusionproteins expressed in yeast.

The activity of each pathway enzyme in whole cell lysates is determinedas described in General Methods. Activity in mitochondrial/organellar(P) and cytosolic (S) fractions and whole cell (W) lysates is assayed asdescribed in General Methods.

Example 8 Adding of Tags, e.g. Ubiquitin Tags, to the N-Terminus of anIsobutanol Pathway Enzyme

The purpose of this is example is to demonstrate that isobutanol pathwayenzymes can be targeted to the yeast cytosol. For instance, this exampleillustrates how a DHAD enzyme can be targeted to the yeast cytosol.

TABLE 18 Genotype of strains disclosed in Example 8. GEVO No.Genotype/Source Gevo2242 S.cerevisiae, CEN.PK; MAT-alpha ura3 leu2 his3trp1 ilv5^(D255E) pdc1::Bs-alsS,TRP1 Gevo2244 S. cerevisiae, CEN.PK;MATα ura3 leu2 his3 trp1 ilv3Δ

TABLE 19 Plasmids disclosed in Example 8. pGV No. Genotype pGV1106 pMB1ori, bla (AmpR), 2 μm ori, URA3, TDH3 promoter- Myc tag-polylinker-CYC1terminator pGV1662 pMB1 ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-(kivD) pGV1784 pUC ori, kanR, Mm_ubiquitin coding sequence pGV1855 pMB1ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-Ll_ilvD pGV1897 pMB1 ori,bla (AmpR), 2 μm ori, URA3, TEF1 promoter- Mm_ubiquitin(Gly-X) pGV1900pMB1 ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter- ScILV3(FL) pGV2019pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter- ScILV3ΔN pGV2052pMB1 ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-Mm_ubiquitin(Gly-X)-ScIlv3(FL) pGV2053 pMB1 ori, bla (AmpR), 2 μm ori,URA3, TEF1 promoter- Mm_ubiquitin(Gly-X)-ScIlv3ΔN pGV2054 pMB1 ori, bla(AmpR), 2 μm ori, URA3, TEF1 promoter- Mm_ubiquitin(Gly-X)-Ll_ilvDpGV2055 pMB1 ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-Mm_ubiquitin(Gly-X)-Gf_ilvD pGV2056 pMB1 ori, bla (AmpR), 2 μm ori,URA3, TEF1 promoter- Mm_ubiquitin(Gly-X)-Se_ilvD

To develop the constructs required to express DHAD as a fusion with anN-terminal ubiquitin, plasmid pGV1784 was synthesized by DNA2.0. Thisplasmid contained the synthesized sequence for the Mus musculusubiquitin gene, codon-optimized for expression in S. cerevisiae (SEQ IDNO: 86). Using this plasmid as the template, the M. musculus ubiquitingene was amplified via PCR using primers 1792 and 1794 to generate a PCRproduct containing the M. musculus ubiquitin gene codon sequence flankedby restriction sites XhoI and NotI at its 5′ and 3′ ends, respectively,and altered so as to lack the codon for its endogenous C-terminal mostglycine residue (denoted as Gly-X). This PCR product was cloned intopGV1662 (described in Example 6), yielding pGV1897.

Plasmid pGV1897 was then used as a recipient cloning vector forsequences encoding S. cerevisiae ILV3 (ScIlv3(FL), SEQ ID NO: 88), S.cerevisiae Ilv3ΔN (ScIlv3ΔN, SEQ ID NO: 89), L. lactis ilvD (LI_ilvD,SEQ ID NO: 87), G. forsetti ilvD (Gf_ilvD, SEQ ID NO: 90), and S.erythraea ilvD (Se_ilvD, SEQ ID NO: 91), yielding plasmids pGV2052-2056,respectively.

The DHAD activity exhibited by cells transformed with each of theresulting constructs is ascertained by in vitro assay. GEVO2244 istransformed (singly) with pGV2052-2056, pGV1106 (empty control vector),pGV1855 (expressing native, unfused LI_ilvD) or pGV1900 (expressingnative, full-length Sc_ILV3(FL)). Lysates of transformants are preparedand DHAD activity in mitochondrial/organellar (P) and cytosolic (S)fractions and whole cell (W) lysates is assayed as described in Example3.

In an analogous manner, a desired ALS (e.g., the B. subtilis alsS) orKARI gene whose product is known or predicted to be mitochondrial can bere-targeted to the cytosol by means of the methods detailed in thisexample. The nucleotide sequence encoding for a full-length, or variant,ALS or KARI is amplified by PCR using primers that introduce restrictionsites convenient for cloning the final product as an in-frame fusion ofthe M. musculus ubiquitin gene. The resulting construct is transformedinto a host S. cerevisiae cell suitable for assaying the in vitroactivity of the expressed M. musculus ubiquitin-gene chimeric fusionprotein, using methods described in Example 3.

Example 9 Dihydroxy Acid Dehydratase Limits Isobutanol Production inYeast

This example illustrates the specific activity of various DHAD homologsin yeast. The example also illustrates that high specific activity ofthe Lactococcus lactis IlvD enzyme (SEQ ID NO: 18) correlates with anincrease in isobutanol production.

Plasmid pGV1106 was used as a control and is described in Example 3.Plasmid pGV1662 (described in Example 6) served as the parental plasmidof pGV1855, pGV1900, and pGV2019 (see Example 5). Plasmids pGV1851-1855and pGV1904-1907 are all variants of pGV1662 (See Table 20), in whichthe kivD ORF sequence present in pGV1662 was excised and replaced with asequence encoding a DHAD homolog, as indicated below.

TABLE 20 Plasmids disclosed in Example 9. pGV No. Genotype pGV1851 pUCori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-Gramella forsetti ilvDpGV1852 pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1promoter-Chromohalobacter salexigens ilvD pGV1853 pUC ori, bla (AmpR), 2μm ori, URA3, TEF1 promoter-Ralstonia eutropha ilvD pGV1854 pUC ori, bla(AmpR), 2 μm ori, URA3, TEF1 promoter-Saccharopolyspora erythraea ilvDpGV1855 pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-Ll_ilvDpGV1900 pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-ScILV3(FL)pGV1904 pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-Acidobacteriabacterium Ellin345 ilvD pGV1905 pUC ori, bla (AmpR), 2 μm ori, URA3,TEF1 promoter-Picrophilus torridus DSM 9790 ilvD pGV1906 pUC ori, bla(AmpR), 2 μm ori, URA3, TEF1 promoter-Piromyces species E2 ilvD pGV1907pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-Sulfolobus tokodaiistrain 7 ilvD

Plasmid pGV1851 contains the G. forsetti ilvD gene (SEQ ID NO: 90).Plasmid pGV1852 contains the C. salexigens gene (SEQ ID NO: 95). PlasmidpGV1853 contains the R. eutropha gene (SEQ ID NO: 94). Plasmid pGV1854contains the S. erythraea ilvD (SEQ ID NO: 91). Plasmid pGV1855 containsthe L. lactis ilvD (SEQ ID NO: 87). Plasmid pGV1900 contains the S.cerevisiae ILV3 (SEQ ID NO: 88). Plasmid pGV1904 contains the A.bacterium Ellin345 ilvD (SEQ ID NO: 92). Plasmid pGV1905 contains the P.torridus DSM 9790 ilvD (SEQ ID NO: 96). Plasmid pGV1906 contains thePiromyces sp. E2 ilvD (SEQ ID NO: 93). Plasmid pGV1907 contains the S.tokodaii ilvD (SEQ ID NO: 97). All sequences (except that of the S.cerevisiae ILV3 (full length) were synthesized with 5′ SalI and 3′ NotIsites by DNA2.0 (Menlo Park, Calif.), digested with SalI and NotI, andligated into pGV1662 which had also been digested with SalI and NotI.For plasmid pGV1900, the sequence containing the open reading frame ofthe S. cerevisiae ILV3 (full length) was amplified from S. cerevisiaegenomic DNA using primers 1617 and 1618, and the resulting 1.8 kbfragment was digested with SalI plus BamHI and cloned into pGV1662.Various DHADs were tested for in vitro activity using whole celllysates. In this case, the DHADs were expressed in a yeast deficient forDHAD activity (GEVO2244; ilv3Δ) (see Example 2) to minimize endogenousbackground activity.

To grow cultures for cell lysates, triplicate independent cultures ofeach desired strain were grown overnight in 3 mL SCD-Ura+9xIV at 30° C.,250 rpm. The following day, the overnight cultures were diluted 1:50into 50 mL fresh SCD-Ura in a 250 mL baffle-bottomed Erlenmeyer flaskand incubated at 30° C. at 250 rpm. After approximately 10 hours, theOD₆₀₀ of all cultures were measured, and the cells of each culture werecollected by centrifugation (2700×g, 5 min). The cell pellets werewashed by resuspending in 1 mL of water, and the suspension was placedin a 1.5 mL tube and the cells were collected by centrifugation(16,000×g, 30 seconds). All supernatant was removed from each tube andthe tubes were frozen at −80° C. until use.

Lysates were prepared by resuspending each cell pellet in 0.7 mL oflysis buffer. Lysate lysis buffer consisted of: 0.1M Tris-HCl pH 8.0, 5mM MgSO₄, with 10 μL of Yeast/Fungal Protease Arrest solution (GBiosciences, catalog #788-333) per 1 mL of lysis buffer. Eight hundredmicroliters of cell suspension were added to 1 mL of 0.5 mm glass beadsthat had been placed in a chilled 1.5 mL tube. Cells were lysed by beadbeating (6 rounds, 1 minute per round, 30 beats per second) with 2minutes chilling on ice in between rounds. The tubes were thencentrifuged (20,000×g, 15 min) to pellet debris and the supernatant(cell lysates) were retained in fresh tubes on ice. The proteinconcentration of each lysate was measured using the BioRad Bradfordprotein assay reagent (BioRad, Hercules, Calif.) according tomanufacturer's instructions.

The DHAD activity of each lysate was ascertained as follows. In a fresh1.5 mL centrifuge tube, 50 μL of each lysate was mixed with 50 μL of0.1M 2,3-dihydroxyisovalerate (DHIV), 25 μL of 0.1 M MgSO₄, and 375 μLof 0.05M Tris-HCl pH 8.0, and the mixture was incubated for 30 min at35° C. Each tube was then heated to 95° C. for 5 min to inactivate anyenzymatic activity, and the solution was centrifuged (16,000×g for 5min) to pellet insoluble debris. To prepare samples for analysis, 100 μLof each reaction were mixed with 100 μL of a solution consisting of 4parts 15 mM dinitrophenyl hydrazine (DNPH) in acetonitrile with 1 part50 mM citric acid, pH 3.0, and the mixture was heated to 70° C. for 30min in a thermocycler. The solution was then analyzed by HPLC asdescribed above in General Methods to quantitate the concentration ofketoisovalerate (KIV) present in the sample. The results are shown inTable 21.

TABLE 21 Specific activities (KIV generation) from lysates of S.cerevisiae strain GEVO2244 carrying plasmids to overexpress theindicated DHAD homolog. Each data point is the result of triplicatesamples. Specific activity Plasmid Gene (U/mg total protein) pGV1106Control (i.e. no DHAD) n.d. pGV1851 Gramella forsetti ilvD 0.012 pGV1852Chromohalobacter salexigens n.d. (SEQ ID NO: 95) pGV1853 Ralstoniaeutropha (SEQ ID NO: 94) n.d. pGV1854 Saccharopolyspora erythraea ilvD0.002 pGV1855 Lactococcus lactis ilvD 0.027 pGV1900 Saccharomycescerevisiae ILV3(FL) 0.148 pGV1904 Acidobacteria bacterium Ellin345 DHAD0.004 pGV1905 Picrophilus torridus DSM 9790 DHAD n.d. pGV1906 PiromycesSp E2 DHAD 0.016 pGV1907 Sulfolobus tokodaii str. 7 DHAD 0.001 * n.d.,not detectable

Example 10 Dihydroxy Acid Dehydratase Limits Isobutanol Production inYeast

This example illustrates that high specific DHAD activity, and inparticular the high specific activity of the L. lactis IlvD enzyme (SEQID NO: 18) correlates with an increase in isobutanol production.

TABLE 22 Genotype of strains disclosed in Example 10. GEVO No.Genotype/Source GEVO1186 S. cerevisiae, CEN.PK; MATa/α ura3/ura3leu2/leu2 his3/his3 trp1/trp1 GEVO1188 S. cerevisiae, CEN.PK; MATα ura3leu2 his3 trp1 GEVO1803 MATa/α ura3/ura3 leu2/leu2 his3/his3 trp1/trp1pdc1::Bs- alsS, TRP1/PDC1 GEVO2107 MATa/α ura3/ura3 leu2/leu2 his3/his3trp1/trp1 pdc1::Bs- alsS, TRP1/PDC1 pdc6::{ScTEF1p-Ll_kivd ScTDH3p-Dm_ADH URA3}/PDC6

TABLE 23 Plasmids disclosed in Example 10. pGV No. Genotype p423GPDP_(TDH3):MCS:T_(CYC1), HIS3, 2-micron, bla, pUC ori (Mumberg, D. et al.(1995) Gene 156: 119-122; obtained from ATCC) pGV1103P_(TDH3):myc-tag:MCS:T_(CYC1), HIS3, 2 micron, bla, pUC ori pGV1730P_(CUP1):Bs-alsS:T_(PDC1)/PDC1-3′ region:PDC1-5′ region, TRP1, bla, pUCori pGV1914 P_(TEF1):Ll_kivD P_(TDH3):Dm_ADH PDC6 5′, 3′ targetinghomology URA3 pUC ori bla(ampR) pGV1974P_(TEF1):Sc_ILV3ΔN:P_(TDH3):Ec_ilvC^(Q110V)-coSc:T_(CYC1), HIS3, 2micron, bla, pUC ori bla(ampR) pGV1981 P_(TEF1):Lactococcus lactisilvD-coSc:P_(TDH3):Ec_ilvC^(Q110V)- coSc:T_(CYC1), HIS3, 2 micron, bla,pUC ori pGV2001 P_(TEF1):P_(TDH3):EC_ilvC^(Q110V)-coSc:T_(CYC1), HIS3, 2micron, bla, pUC ori

Plasmid pGV1103 was generated by inserting a linker (primers 271annealed to primer 272) containing a myc-tag and a new MCS(SalI-EcoRI-SmaI-BamHI-NotI) into the SpeI and XhoI sites of p423GPD.The construction of plasmid pGV1730 is described in Example 4.

pGV1914 (SEQ ID NO: 117) is a yeast integrating vector that includes theS. cerevisiae URA3 gene as a selection marker and contains homologoussequence for targeting the HpaI-digested, linearized plasmid forintegration at the PDC6 locus of S. cerevisiae. pGV1914 carries the D.melanogaster adh (Dm_ADH) (SEQ ID NO: 116) and L. lactis kivd (LI_kivD)genes, expressed under the control of the S. cerevisiae TDH3 and TEF1promoters, respectively. The open reading frame sequence of DmADH wasoriginally amplified by PCR from clone RH54514 (available from theDrosophila Genome Resource Center).

Plasmid pGV1974 is a yeast high copy plasmid with HIS3 as a marker forthe expression of E. coli ilvC^(Q110V) (SEQ ID NO: 98) and S. cerevisiaeILV3ΔN (SEQ ID NO: 89). pGV1974 was generated by cloning a SacI-NotIfragment (4.9 kb, SEQ ID NO: 118) carrying the S. cerevisiae TEF1promoter:S. cerevisiae ilv3ΔN:S. cerevisiae TDH3 promoter:E. coliilvC^(Q110V) into the SacI-NotI sites of pGV1103 (5.4 kb), a yeastexpression plasmid carrying the HIS3 marker.

Plasmid pGV1981 is a yeast high copy plasmid with HIS3 as a marker forthe expression of E. coli ilvC^(Q110V) and L. lactis ilvD. pGV1981 wasgenerated by cloning a SalI-BamHI fragment (1.7 kb) carrying the L.lactis ilvD ORF (SEQ ID NO: 87 with a SalI and BamHI sites introduces atthe 5′ and 3′ ends, respectively) into the SalI-BamHI of pGV1974 (8.5kb), replacing the S. cerevisiae Ilv3ΔN ORF.

Plasmid pGV2001 is a yeast high copy plasmid with HIS3 as a marker forthe expression of E. coli ilvC^(Q110V). pGV2001 was generated bydigesting pGV1974 with SalI-BamHI to remove the S. cerevisiae Ilv3ΔNORF. The digest was treated with Klenow to fill-in the 5′ overhangs, thelarger 8.5 kb fragment was isolated and self-ligated.

GEVO1803 was made by transforming GEVO1186 with the 6.7 kb pGV1730(contains S. cerevisiae TRP1 marker and the CUP1 promoter-driven B.subtilis alsS) that had been linearized by digestion with NruI.Completion of the digest was confirmed by running a small sample on agel. The digested DNA was then purified using Zymo Research DNA Cleanand Concentrator and used in the transformation. Trp+ clones wereconfirmed for the correct integration into the PDC1 locus by colony PCRusing primer pairs 1440+1441 and 1442+1443 for the 5′ and 3′ junctions,respectively. Expression of B. subtilis alsS was confirmed by qRT-PCRusing primer pairs 1323+1324.

GEVO2107 was made by transforming GEVO1803 with linearized,HpaI-digested pGV1914. Correct integration of pGV1914 at the PDC6 locuswas confirmed by analyzing candidate Ura+ colonies by colony PCR usingprimers 1440 plus 1441, or 1443 plus 1633, to detect the 5′ and 3′junctions of the integrated construct, respectively. Expression of alltransgenes were confirmed by qRT-PCR using primer pairs 1321 plus 1322,1587 plus 1588, and 1633 plus 1634 to examine Bs_alsS, LI_kivD, andDm_ADH transcript levels, respectively.

GEVO 2107 was transformed with plasmids that contained either a KARIalone (pGV2001 with E. coli ilvC^(Q110V)) or the same KARI with a DHAD(pGV1974 with the S. cerevisiae Ilv3ΔN or pGV1981 with the L. lactisilvD). Fermentations were carried out with three independenttransformants for each DHAD homolog being tested, as well as the no DHADcontrol plasmid. Seed cultures were grown in SCD-H medium to mid-logphase. The fermentations were initiated by collecting cells andresuspending in 25 mL of SCD-H (5% glucose) medium to an OD₆₀₀ of 1.Fermentations were performed aerobically in 125 mL unbaffled flasksshaken at 250 rpm at 30° C. At 0, 24, 48 and 72 hours, the OD₆₀₀ waschecked and 2 mL samples were taken. These samples were centrifuged at18,000×g in a microcentrifuge and 1.5 mL of the clarified media wastransferred to a 1.5 mL Eppendorf tube. The clarified media was storedat 4° C. until analyzed by GC and HPLC as described in General Methods.At 24 and 48 hours, 2.5 mL of glucose from a 400 g/L stock solution wasadded to the cultures. FIG. 4 shows the production of isobutanol inthese fermentations. All values were adjusted for the dilution caused bythe volume change from adding glucose. An increased amount of isobutanolwas produced from the cells expressing the L. lactis ilvD.

Example 11 Assaying DHAD Activity in Fractionated Cell Extracts

The purpose of this Example is to describe how DHAD activity can bemeasured in fractionated cellular extracts that are enriched for eithermitochondrial or soluble cytosolic components.

Plasmids pGV1106, pGV1662, pGV1855, pGV1900 are described in Example 9above. To measure the DHAD activities present in fractionated cellextracts, the strain GEVO2244 was transformed singly with eitherpGV1106, which served as an empty vector control, or with one of:pGV1855, pGV1900, or pGV2019, which are expression plasmids for L.lactis ilvD, S. cerevisiae ILV3 (full length), and S. cerevisiae ILV3ΔN,respectively.

An independent clonal transformant of each plasmid was isolated, and a 1L culture of each strain was grown in SCGaI-Ura+9xIV at 30° C. at 250rpm. The OD₆₀₀ was noted, the cells were collected by centrifugation(1600×g, 2 min) and the culture medium was decanted. The cell pelletswere resuspended in 50 mL sterile deionized water, collected bycentrifugation (1600×g, 2 min), and the supernatant was discarded. TheOD₆₀₀ and total wet cell pellet weight of each culture are listed inTable 24, below:

TABLE 24 OD₆₀₀ and pellet mass (g) of strain GEVO2244 transformed withthe indicated plasmids. Pellet mass Plasmid OD₆₀₀ (g) pGV1106 2.2 7.6pGV1855 2.3 7.7 pGV1900 1.3 3.8 pGV2019 2.6 8.4

To obtain spheroplasts, the cell pellets were resuspended in 0.1 MTris-SO₄, pH 9.3, to a final concentration of 0.1 g/mL, and DTT wasadded to a final concentration of 10 mM. Cells were incubated withgentle (60 rev/min) agitation on an orbital shaker for 20 min at 30° C.,and the cells were then collect by centrifugation (1600×g, 2 min) andthe supernatant discarded. Each cell pellet was resuspended inspheroplasting buffer, which consists of (final concentrations): 1.2Msorbitol (Amresco, catalog #0691), 20 mM potassium phosphate pH 7.4) andthen collected by centrifugation (1600×g, 10 min). Each cell pellet wasresuspended in spheroplasting buffer to a final concentration of 0.1 gcells/mL in a 500 mL centrifuge bottle, and 50 mg of Zymolyase 20T(Seikagaku Biobusiness, Code#120491) was added to each cell suspension.The suspensions were incubated overnight (approximately 16 hrs) at 30°C. with gentle agitation (60 rev/min) on an orbital shaker. The efficacyof spheroplasting was ascertained by diluting an aliquot of each cellsuspension 1:10 in either sterile water or in spheroplasting buffer, andcomparing the aliquots microscopically (under 40× magnification). In allcases, >90% of the water-diluted cells lysed, indicating efficientspheroplasting. The spheroplasts were centrifuged (3000×g, 10 min, 20°C.), and the supernatant was discarded. Each cell pellet was resuspendedin 50 mL spheroplast buffer without Zymolyase, and cells were collectedby centrifugation (3000×g, 10 min, 20° C.).

To fractionate spheroplasts, the cells were resuspended to a finalconcentration of 0.5 g/mL in ice cold mitochondrial isolation buffer(MIB), consisting of (final concentration): 0.6M D-mannitol (BD DifcoCat#217020), 20 mM HEPES-KOH, pH 7.4. For each 1 mL of resulting cellsuspension, 0.01 mL of Yeast/Fungal Protease Arrest solution (GBiosciences, catalog #788-333) was added. The cell suspension wassubjected to 35 strokes of a Dounce homogenizer with the B (tight)pestle, and the resulting cell suspension was centrifuged (2500×g, 10min, 4° C.) to collect cell debris and unbroken cells and spheroplasts.Following centrifugation, 2 mL of each sample (1 mL of the pGV1900transformed cells) were saved in a 2 mL centrifuge tube on ice anddesignated the “W” (for Whole cell extract) fraction, while theremaining supernatant was transferred to a clean, ice-cold 35 mLOakridge screw-cap tube and centrifuged (12,000×g, 20 min, 4° C.) topellet mitochondria and other organellar structures. Followingcentrifugation, 5 mL of each resulting supernatant was transferred to aclean tube on ice, being careful to avoid the small, loose pellet, andlabelled the “S” (soluble cytosol) fraction. The resulting pellets wereresuspended in MIB containing Protease Arrest solution, and werelabelled the “P” (“pellet”) fractions. Protein from the “P” fraction wasreleased after dilution 1:5 in DHAD assay buffer (see above) by rapidmixing in a 1.5 mL tube with a Retsch Ball Mill MM301 in the presence of0.1 mM glass beads. The bead-beating was performed 4 times for 1 minute,30 beats per second, after which insoluble debris was removed bycentrifugation (20,000×g, 10 min, 4° C.) and the soluble portionretained for use.

The BioRad Protein Assay reagant (BioRad, Hercules, Calif.) was usedaccording to manufacturer's instructions to determine the proteinconcentration of each fraction; the data are summarized in Table 25,below:

TABLE 25 Protein concentrations of mitochondrial/organellar (P) andcytosolic (S) fractions and whole cell (W) lysates, prepared asdescribed in the text. plasmid/fraction protein [μg/pL] 1106 P 20.3 1855P 17.7 1900 P 9.2 2019 P 19.7 1106 S 12.3 1855 S 12.9 1900 S 7.9 2019 S12.4 1106 W 14.0 1855 W 15.0 1900 W 7.9 2019 W 14.7

The DHAD activity of each fraction was ascertained as follows. In afresh 1.5 mL centrifuge tube, 50 μL of each fraction was mixed with 50μL of 0.1M 2,3-dihydroxyisovalerate (DHIV), 25 μL of 0.1 M MgSO₄, and375 μL of 0.05M Tris-HCl pH 8.0, and the mixture was incubated for 30min at 35° C. Each reaction was carried out in triplicate. Each tube wasthen heated to 95° C. for 5 min to inactivate any enzymatic activity,and the solution was centrifuged (16,000×g for 5 min) to pelletinsoluble debris. To prepare samples for analysis, 100 μL of eachreaction were mixed with 100 μL of a solution consisting of 4 parts 15mM dinitrophenyl hydrazine (DNPH) in acetonitrile with 1 part 50 mMcitric acid, pH 3.0, and the mixture was heated to 70° C. for 30 min ina thermocycler. Analysis of ketoisovalerate via HPLC was carried out asdescribed in General Methods. Data from the experiment are summarizedbelow in Table 26.

TABLE 26 Specific activities (KIV generation) and ratios of specificactivities from fractionated lysates of S. cerevisiae strain GEVO2244carrying plasmids to overexpress the indicated DHAD homolog. Each datapoint is the result of triplicate samples. Sp. Activity Ratio of Sp.Lysate [U/mg Activities (pGV# and protein in (Cyto or Mito fraction*)DHAD fraction] Std. Dev. to Whole-Cell) 1106 WCL — n.d. 1106 cyto — n.d.1106 mito — n.d. 1855 WCL Ll_ilvD 0.0006 4.7E−05 1855 cyto Ll_ilvD0.0011 0.0001 1.76 1855 mito Ll_ilvD 2E−05 3.5E−05 0.03 1900 WCLScILV3(FL) 0.0096 0.0018 1900 cyto ScILV3(FL) 0.0052 0.0004 0.54 1900mito ScILV3(FL) 0.0340 0.0029 3.53 *WCL, whole cell lysate; cyto,cytosolic-enriched fraction; mito, mitochondrial (organellar)-enrichedfraction

Cells overexpressing the L. lactis ilvD generated a significantlygreater proportion of DHAD activity in the cytosolic fraction versus themitochondrial fraction, whereas cells overexpressing the full-length,native (mitochondrial) S. cerevisiae ILV3 resulted in a greaterproportion of the specific activity residing in the mitochondrialfraction.

Example 12 Alternative, Native Dehydratases with DHAD Activity

This example describes how the overexpression of native dehydratases inS. cerevisiae for the conversion of 2,3-dihydroxyisovalerate toketoisovalerate is measured.

TABLE 27 Plasmids disclosed in Example 12. pGV No. Genotype p426TEFP_(TEF1):MCS:T_(CYC1), URA3, 2-micron, bla, pUC-ori (Mumberg, D. et al.(1995) Gene 156: 119-122; obtained from ATCC) 1102P_(TEF1):HA-tag:MCS:T_(CYC1), URA3, 2-micron, bla, pUC-ori 1106P_(TDH3):myc-tag:MCS:T_(CYC1), URA3, 2-micron, bla, pUC-ori 1662P_(TEF1):Ll_kivd: T_(CYC1), URA3, 2-micron, bla, pUC-ori 1894P_(TEF1):Ec_ilvC^(Q110V)-coSc:T_(CYC1), URA3, 2-micron, bla, pUC-ori2000 P_(TEF1):Sc_ILV3ΔN: P_(TDH3):Ec_ilvC^(Q110V)-coSc: T_(CYC1), URA3,2-micron, bla, pUC-ori 2111 P_(TEF1):Ll_ilvD:P_(TDH3):Ec_ilvC^(Q110v)-coSc:T_(CYC1), URA3, 2- micron, bla,pUC-ori 2112 P_(TEF1):Sc_LEU1:P_(TDH3):Ec_ilvC^(Q110V)-coSc:T_(CYC1),URA3, 2-micron, bla, pUC-ori 2113P_(TEF1):Sc_HIS3:P_(TDH3):Ec_ilvC^(Q110V)-coSc:T_(CYC1), URA3, 2-micron,bla, pUC-ori

Plasmid pGV1102 was generated by inserting a linker (primers 269annealed to primer 270) containing a HA-tag and a new MCS(SalI-EcoRI-SmaI-BamHI-NotI) into the SpeI and XhoI sites of p426TEF.Plasmids pGV1106 and pGV1662 are described in Examples 3 and 5,respectively. Plasmid pGV1894 is a yeast high copy plasmid with URA3 asa marker for the expression of E. coli ilvC^(Q110V) and was generated bycloning a XhoI-NotI fragment (1.5 kb) carrying the E. coli ilvC^(Q110V)ORF (SEQ ID NO: 98) into the SalI-NotI of pGV1662 (6.3 kb), replacingthe L. lactis kivD ORF. Plasmids pGV2000, pGV2111, pGV2112, and pGV2113are yeast high copy plasmids with URA3 as a marker for the expression ofE. coli ilvC^(Q110V) and a DHAD. pGV2000 is generated by cloning aSacI-NotI fragment (4.9 kb) from pGV1974 (described in Example 10)carrying the S. cerevisiae TEF1 promoter:S. cerevisiae Ilv3ΔN:S.cerevisiae TDH3 promoter:E. coli ilvC^(Q110V) into the SacI-NotI sitesof pGV1106 (6.6 kb), a yeast expression plasmid carrying the URA3marker. pGV2111 is generated by cloning a SalI-BamHI fragment (1.7 kb)carrying the L. lactis ilvD ORF (SEQ ID NO: 97 with SalI and BamHI sitesintroduced at the 5′ and 3′ ends, respectively) into the SalI-BamHI ofpGV2000 (8.4 kb), replacing the S. cerevisiae Ilv3ΔN ORF. pGV2112 isgenerated by cloning the S. cerevisiae LEU1 gene as a SalI-BamHIfragment (2.3 kb), generated by PCR using primers 2163 and 1842 usinggenomic DNA as template, into the SalI-BamHI of pGV2000 (8.4 kb),replacing the S. cerevisiae Ilv3ΔN ORF. pGV2113 is generated by cloningthe S. cerevisiae HIS3 gene as a SalI-BamHI fragment (0.7 kb), generatedby PCR using primers 2183 and 2184 using genomic DNA as template, intothe SalI-BamHI of pGV2000 (8.4 kb), replacing the S. cerevisiae Ilv3ΔNORF.

DHADs are tested for in vitro activity using whole cell lysates. TheDHADs as well as LEU1 and HIS3 are expressed from pGV2000, pGV2112, andpGV2113 GEVO2244 to minimize endogenous DHAD background activity. Aplasmid that does not express DHAD, pGV1894, and a plasmid thatexpresses the L. lactis ilvD, pGV2111, are used as negative and positivecontrols, respectively

To grow cultures for cell lysates, triplicate independent cultures ofeach desired strain are grown overnight in 3 mL YNBD+HLW+10xIV at 30°C., 250 rpm. The following day, the overnight cultures are diluted 1:50into 50 mL fresh YNBD+HLW+10xIV in a 250 mL baffle-bottomed Erlenmeyerflask and incubated at 30° C. at 250 rpm. After approximately 10 hours,the OD₆₀₀ of all cultures are measured, and the cells of each cultureare collected by centrifugation (2700×g, 5 min). The cell pellets arewashed by resuspending in 1 mL of water, and the suspension is placed ina 1.5 mL tube and the cells are collected by centrifugation (16,000×g,30 seconds). All supernatant is removed from each tube and the tubes arefrozen at −80° C. until use.

Lysates are prepared by resuspending each cell pellet in 0.7 mL of lysisbuffer. Lysate lysis buffer consisted of: 0.1M Tris-HCl pH 8.0, 5 mMMgSO₄, with 10 μL of Yeast/Fungal Protease Arrest solution (GBiosciences, catalog #788-333) per 1 mL of lysis buffer. Eight hundredmicroliters of cell suspension are added to 1 mL of 0.5 mm glass beadsthat had been placed in a chilled 1.5 mL tube. Cells are lysed by beadbeating (6 rounds, 1 minute per round, 30 beats per second) with 2minutes chilling on ice in between rounds. The tubes are thencentrifuged (20,000×g, 15 min) to pellet debris and the supernatant(cell lysates) are retained in fresh tubes on ice. The proteinconcentration of each lysate is measured using the BioRad Bradfordprotein assay reagent (BioRad, Hercules, Calif.) according tomanufacturer's instructions.

The DHAD activity of each lysate is ascertained as follows. In a fresh1.5 mL centrifuge tube, 50 μL of each lysate is mixed with 50 μL of 0.1M2,3-dihydroxyisovalerate (DHIV), 25 μL of 0.1 M MgSO₄, and 375 μL of0.05M Tris-HCl pH 8.0, and the mixture is incubated for 30 min at 35° C.Each tube is then heated to 95° C. for 5 min to inactivate any enzymaticactivity, and the solution is centrifuged (16,000×g for 5 min) to pelletinsoluble debris. To prepare samples for analysis, 100 μL of eachreaction are mixed with 100 μL of a solution consisting of 4 parts 15 mMdinitrophenyl hydrazine (DNPH) in acetonitrile with 1 part 50 mM citricacid, pH 3.0, and the mixture is heated to 70° C. for 30 min in athermocycler. The solution is then analyzed by HPLC as described abovein General Methods to quantitate the concentration of ketoisovalerate(KIV) present in the sample.

DHADs are tested for in vitro activity using whole cell lysates. TheDHADs are expressed in a yeast deficient for DHAD activity (GEVO2244;ilv3Δ) to minimize endogenous background activity.

Example 13 Cloning of Low-Abundance, Endogenous Cytosolic Iron-SulfurCluster Assembly Machinery for Overexpression in S. cerevisiae

The purpose of this example is to describe how three known components ofthe S. cerevisiae cytosolic iron-sulfur assembly machinery were clonedto permit their overexpression in S. cerevisiae, to increase cytosolicDHAD activity.

In the yeast S. cerevisiae, at four least genes—CIA1, CFD1, NAR1, andNBP35—encode activities that contribute to the proper assembly and/ortransfer of iron-sulfur [Fe—S] clusters of cytosolic proteins. Of thesefour genes, three—CFD1, NAR1, and NBP35—have been shown to be expressedat very low levels during aerobic growth on glucose (Ghaemmaghami etal., 2003, Nature, 425: 737-741). These three genes thus representattractive candidates for overexpression to increase the cellularcapacity for proper cytosolic [Fe—S] cluster protein assembly.

TABLE 27 Plasmids disclosed in Example 13. pGV No. Genotype pGV2074 pUCori, bla (AmpR), 2 μm ori, TPI1 promoter-hph (HygroR),PGK1 promoter,TEF1 promoter, TDH3 promoter pGV2127 pUC ori, bla (AmpR), 2 μm ori, TPI1promoter-hph (HygroR), PGK1 promoter, TEF1 promoter, TDH3 promoter-CFD1pGV2138 pUC ori, bla (AmpR), 2 μm ori, TPI1 promoter-hph (HygroR), PGK1promoter, TEF1 promoter-NAR1, TDH3 promoter-CFD1 pGV2144 pUC ori, bla(AmpR), 2 μm ori, TPI1 promoter-hph (HygroR), PGK1 promoter- NBP35, TEF1promoter, TDH3 promoter pGV2147 pUC ori, bla (AmpR), 2 μm ori, TPI1promoter-hph (HygroR), PGK1 promoter- NBP35, TEF1 promoter-NAR1, TDH3promoter-CFD1

To clone the sequences for CFD1, NAR1, and NBP35 into an appropriate S.cerevisiae expression vector, the following steps were carried out:Vector pGV2074 (SEQ ID NO: 133) was used as a parental plasmid forsubsequent cloning steps described below. The salient features ofpGV2074 include a bacterial origin of replication (pUC) and selectablemarker (bla), an S. cerevisiae 2 μm origin of replication and selectablemarker (the hph gene, conferring resistance to hygromycin, operablylinked to the TPI1 promoter region), and sequences containing the S.cerevisiae promoters for the PGK1, TDH3 and TEF1 genes, each followed byone or more unique restriction sites to facilitate the introduction ofcoding sequences.

First, the CFD1 coding sequence was amplified from S. cerevisiae genomicDNA by PCR, using primers 2195 and 2196, which also added 5′ XhoI and 3′NotI sites, respectively. The resulting ˜890 bp product was digestedwith XhoI plus NotI and ligated into pGV2074 that had been digested withXhoI plus NotI, yielding the plasmid pGV2127. All sequences amplified byPCR were confirmed by DNA sequencing. Next, the NAR1 coding sequence wasamplified from S. cerevisiae genomic DNA by PCR, using primers 2197 and2198, which added 5′ SalI and 3′ BamHI sites, respectively. Theresulting ˜1485 bp product was digested with SalI plus BamHI and clonedinto pGV2127 which had also been digested with SalI plus BamHI, therebyyielding pGV2138. Next, the NBP35 coding sequence was amplified S.cerevisiae genomic DNA by PCR, using primers 2259 and 2260, which added5′ BglII and 3′ KpnI and XhoI (from 5′ to 3′) sites, respectively. Theresulting ˜995 bp product was digested with BglII plus XhoI and ligatedinto pGV2074 that had been digested with BglII plus SalI, yieldingpGV2144. Finally, pGV2144 was digested with AvrII plus BamHI, and theresulting 1.78 kb fragment (which contained the PGK1 promoter and theNBP35 ORF sequence) was gel purified and ligated into the vector pGV2138that had been digested with AvrII plus BglII, yielding pGV2147.

Example 14 Cloning of Heterologous Cytosolic Iron-Sulfur ClusterAssembly Machinery for Overexpression in S. cerevisiae

The purpose of this example is to describe how one or more cytosoliciron-sulfur assembly machinery components, from various species, can becloned to permit their overexpression in S. cerevisiae, therebyincreasing cytosolic DHAD activity.

In addition to the endogenous cytosolic iron-sulfur assembly machineryfound in S. cerevisiae, homologous sequences and activities have beenidentified in other microbial and eukaryotic species. In one example,the ApbC protein of Salmonella enterica serovar Typhimurium has beenshown, in vitro, to bind and effectively transfer iron-sulfur clustersto a known cytosolic [Fe—S] cluster-containing S. cerevisiae substrate,Leu1 (Boyd et al., 2008, Biochemistry, 47: 8195-202). Thus, a number ofother useful homologs of the known S. cerevisiae cytosolic iron-sulfurassembly machinery components exist and present attractive candidatesfor overexpression in S. cerevisiae. Table 28 lists several exemplaryhomologs and their GenBank accession numbers, as identified by previoushomology searches (Boyd et al., 2009, J. Biol Chem 284: 110-118). Alsoincluded in the table are two closely related S. cerevisiae homologs,Nbp35 and Cfd1. Of note, Ind1 is reported to be localized to andfunctional in the mitochondria (Bych et al., 2008, EMBO J. 27: 1736-46),whereas Hcf101 is reported to participate in iron-sulfur clusterassembly in Arabidopsis chloroplasts (Lezhneva et al., 2004, Plant J.Cell Mol Biol 37: 174-185).

TABLE 28 Functionally homologous proteins involved in iron-sulfurcluster formation. Gene Source, Accession Number ApbC Salmonellaenterica serovar Typhimurium LT2, NP_461098 Ind1 Yarrowia lypolytica,YALI0B18590g Hcf101 Arabidopsis thaliana, AAR97892.1 Nubp1 Homo sapiens,NP_002475.2 Nbp35 S. cerevisiae, CAA96797.1 Cfd1 S. cerevisiae, AAS56623

The cloning of one or more of these genes is carried out usingtechniques well known to one skilled in the art. Oligonucleotide primersare designed that are homologous to the 5′ and 3′ ends of each desiredreading, and which furthermore incorporate a restriction site sequenceconvenient for the cloning of each reading frame into vector pGV2074. Astandard PCR reaction is used to amplify each gene, either from thegenome of each host organism, or from an in vitro synthesized DNAfragment, and the resulting PCR product is cloned into an expressionvector (pGV2074). In the case of a protein known to be targeted to themitochondria, such as Yarrowia lypolytica Ind1, PCR primers are designedto amplify the majority of the coding sequence while excluding the knownN-terminal mitochondrial targeting sequence (Bych et al., 2008, EMBO J.27: 1736-46).

Example 15 Overexpression of S. cerevisiae Cytosolic Iron-SulfurAssembly Machinery to Increase Cytosolic DHAD Activity

The purpose of this example is to describe how a plasmid expressing oneor more iron-sulfur assembly machinery components is co-expressed with aDHAD, thereby increasing the cytosolic activity of the DHAD.

Strain GEVO2244 is simultaneously co-transformed with one of: pGV1851,pGV1852, pGV1853, pGV1854, pGV1855, pGV1904, pGV1905, pGV1906, orpGV1907 (pGV1851-55 and pGV1904-07 are described in Table 20); plus, oneof either: pGV2074 (Table 27) (which serves as an empty-vector control)or pGV2147 (Table 27) (which serves as the cytosolic Fe—S clustermachinery overexpression plasmid), and doubly-transformed cells areselected by plating onto SCD-Ura+9xIV containing 0.1 g/L Hygromycin B.

Three independent isolates from each transformation are cultured inSCD-Ura+9xIV containing 0.1 g/L Hygromycin B to obtain a cell masssuitable for preparation of a lysate, as described in Example 3. Lysatesare prepared from each culture, and the resulting lysates are assayedfor DHAD activity as described in Example 3. To further confirm that theincreased DHAD activity is due specifically to increased cytosolicactivity, cultures of GEVO2244 containing pGV1855 plus either pGV2074 orpGV2147 are grown in SCD-Ura+9xIV containing 0.1 g/L Hygromycin B asotherwise described in Example 11. Fractionated lysates are prepared andin vitro assays to measure DHAD activity are further carried out asdescribed in Example 11.

Example 16 Deletion of LEU1

The purpose of this example is to describe the deletion of LEU1 toincrease the iron-sulfur cluster availability in the yeast cytosol.

TABLE 29 Plasmids disclosed in Example 16. pGV No. Genotype pGV1299 K.lactis URA3, bla, pUC-ori (GEVO) pGV1981 P_(TEF1):Lactococcus lactisilvD-coSc:P_(TDH3):Ec_ilvC^(Q110V)- coSc:T_(CYC1), HIS3, 2-micron, bla,pUC-ori pGV2001 P_(TEF1):P_(TDH3):Ec_ilvC^(Q110V)-coSc:T_(CYC1), HIS3,2-micron, bla, pUC-ori

The LEU1 gene was deleted by transforming cells with a leu1:K. lactisURA3 deletion cassette that was generated by two rounds of PCR.Initially, the K. lactis URA3 gene was amplified with primers 2171 and2172 from pGV1299 (described in Example 2). These primers add 40 bp ofthe LEU1 promoter and terminator sequences to the 5′ and 3′ ends of theK. lactis URA3 gene. This PCR product was then used as a template for aPCR using primers 2170 and 2173. Primer 2170 adds an additional 36 bp ofthe LEU1 promoter sequence at the 5′ end and primer 2173 adds anadditional 38 bp of the LEU1 terminator sequence at the 3′ end. This PCRproduct was transformed into GEVO2244 (described in Example 2) togenerate GEVO2570. The 5′ junction of the integrations were confirmed bycolony PCR using primers 2226 and 587. The 3′ junction of theintegrations were confirmed by colony PCR using primers 588 and 2175.The loss of the LEU1 gene was confirmed by a lack of PCR product usingprimers 2167 and 2227.

GEVO2570 has a deletion in ILV3. GEVO2570 is used to measure DHADactivity in the presence of L. lactis ilvD overexpressed as described inExamples 2 and 4. A plasmid (pGV2001) with no DHAD is used as a negativecontrol.

Example 17 Conserved Motif Amongst Cytosolically Active DHAD Enzymes

This example illustrates that a DHAD enzymes with a specific amino acidsequence motif are more likely to be functional when expressed in theyeast cytosol.

Based on the data from biochemical assays (see Example 10), several DHADhomologs were identified that exhibit at least some cytosolic activity.A total of ten different homologs were tested using biochemical assays.The DHADs were expressed from 2 micron yeast vectors and transformedinto GEVO2244. The homologs were then ranked based on their measuredspecific activity in both whole cell lysates and in cytosolic fractions.

Based on these data, four DHAD homologs: L. lactis (SEQ ID NO: 18), G.forsetii (SEQ ID NO: 17), Acidobacteria (SEQ ID NO: 16), and S.erythraea (SEQ ID NO: 19) exhibit cytosolic DHAD activity. Four DHADhomologs exhibit no cytosolic DHAD activity: R. eutropha (SEQ ID NO:22), C. salexigens (SEQ ID NO: 23), P. torridus (SEQ ID NO: 24), and S.tokodaii (SEQ ID NO: 25). One motif-containing homolog was inconclusive:Piromyces sp. E2 (SEQ ID NO: 21), which did not complement the GEVO2242valine auxotrophy and had detectable biochemical DHAD activity. Since,this homolog has a putative organellar targeting sequence, the proteinis likely to be mitochondrially located explaining its inability tocomplement the GEVO2242 auxotrophy, despite containing the motif.

A multiple sequence alignment (MSA) was created using the Align MultipleSequences tool of Clone Manager 9 Professional Addition Software usingthe “MultiWay” function. This function performs exhaustive pairwiseglobal alignments of all sequences and progressive assembly ofalignments using Neighbor-Joining phylogeny. A total of 53representative DHAD homologs (FIG. 5) were aligned using the followingusing the BLOSUM62 scoring matrix setting. This alignment generated thetree in FIG. 5.

Many of the DHAD homologs exhibiting cytosolic activity are related byoverall homology (>40%) homology when compared to the S. cerevisiae DHADencoded by S. cerevisiae ILV3 (e.g. L. lactis, G. forsetii,Acidobacteria, and S. erythraea). However, the 40% homology cut-offstill includes several DHAD homologs that do not exhibit cytosolic DHADactivity (e.g. R. eutropha, C. salexigens, P. torridus, and S.tokodaii). The Piromyces sp. E2 DHAD failed to complement in thegenetic/biochemistry assay but this result is still consistent with ourmotif hypothesis since the protein still retained its mitochondriallocalization signal. Therefore, a common sequence motif, unique to DHADhomologs that are cytosolically active, was identified:P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27), where (I/L) indicates an isoleucineor leucine at that position, and X indicates any natural or non-naturalamino acid. This motif can be found in all DHAD homologs exhibitingcytosolically activity. Furthermore, an even more specific version ofthis motif was identified that is conserved in all of DHAD homologsexhibiting cytosolic activity except for the S. erythraea DHAD:PIKXXGX(I/L)XIL (SEQ ID NO: 28). This motif is conserved amongst themajority if not all eukaryotic homologs of DHAD.

Six additional DHAD homologs were identified: SEQ ID NOs: 10-15 asspecified in Table 1. These DHAD homologs (SEQ ID NOs: 10-15) containthe motifs PYHKEGGLGIL (SEQ ID NO: 145), PYSEKGGLAIL (SEQ ID NO: 146),PYKPEGGIAIL (SEQ ID NO: 147), PLKPSGHLQIL (SEQ ID NO: 148), PIKKTGHLQIL(SEQ ID NO: 149), and PIKETGHIQIL (SEQ ID NO: 150), respectively.

Example 18 Use of Cytosolically Localized DHADs for the Production ofIsobutanol

The following example illustrates the use of DHADs that have cytosolicactivity in yeast and when expressed in the context of an isobutanolbiosynthetic pathway lead to isobutanol production.

A yeast strain that contains one integrated copy of the B. subtilis alsSgene codon-optimized for expression in S. cerevisiae (SEQ ID NO: 144),two integrated copies of the L. lactis kivD gene (SEQ ID NOs: 99 and151), one integrated copy of L. lactis adhA^(RE1) gene (SEQ ID NO: 152),and one integrated copy of the S. cerevisiae AFT1 gene (SEQ ID NO: 153)was transformed with high copy three-component isobutanol pathwayplasmids containing a KARI (Ec_ilvC_coSc^(P2D1-A1-his6), SEQ ID NO:154), an ADH (L. lactis adhA^(RE1), SEQ ID NO: 152) and a DHAD which wasexpressed from the S. cerevisiae PDC1-286 promoter. The DHAD variedaccording to Table 31. Isobutanol titer and DHAD activity of each strainwas compared to that of a control strain that did not express a DHAD inthe plasmid. Strains, plasmids, and DHADs are listed in Tables 30, 31,and 32, respectively.

TABLE 30 Genotype of strains disclosed in Example 18. GEVO No. GenotypeGEVO3868 S. cerevisiae, CEN.PK2, MATa ura3 leu2 his3 trp1gpd1::T_(Kl)_URA3 gpd2::T_(Kl)_URA3 tma29::T_(Kl)_URA3pdc1::P_(PDC1)-Ll_kivD2_coSc5-P_(FBA1)-LEU2-T_(LEU2)-P_(ADH1)-Bs_alsS1_coSc-T_(CYC1)-P_(PGK1)-Ll_kivD2_coEc-P_(ENO2)-Sp_HIS5 pdc5::T_(Kl)_URA3pdc6::P_(TDH3)-Sc_AFT1-P_(ENO2)-Ll_adhA^(RE1)-T-_(Kl)_URA3_short-P_(FBA1)-Kl_URA3-T_(Kl)_URA3 {evolvedfor C2 supplement-independence, glucose tolerance and faster growth}

TABLE 31 Plasmids disclosed in Example 18. Plasmid Name DHAD GenotypepGV2663 none P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6),P_(ENO2)-Ll_adhA^(RE1), 2 μ-ori, pUC ori, bla, G418r pGV2635 L. lactisP_(PDC1-286)-Ll_ilvD_coSc, P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6),P_(ENO2)-Ll_adhA^(RE1), 2 μ-ori, pUC ori, bla, G418r pGV2671 S.cerevisiae P_(PDC1-286)-Sc_ilv3_ΔN20,P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6), P_(ENO2)-Ll_adhA^(RE1,) 2 μ-ori,pUC ori, bla, G418r pGV2672 G. forsetii P_(PDC1-286)-Gf_ilvD_coSc,P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6), P_(ENO2)-Ll_adhA^(RE1,) 2 μ-ori,pUC ori, bla, G418r pGV2673 S. erythraea P_(PDC1-286)-Se_ilvD_coSc,P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6), P_(ENO2)-Ll_adhA^(RE1,) 2 μ-ori,pUC ori, bla, G418r pGV2674 F. tularensis P_(PDC1-286)-Ft_ilvD_coSc,P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6), P_(ENO2)-Ll_adhA^(RE1) 2 μ-ori,pUC ori, bla, G418r pGV2675 S. cerevisiae P_(PDC1-286)-Sc_ilv3_ΔN19,ilv3ΔN19 P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6), P_(ENO2)-Ll_adhA^(RE1) 2μ-ori, pUC ori, bla, G418r pGV2676 S. P_(PDC1-286)-Sc_ilv3_ΔN23,cerevisiae P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6), ilv3ΔN23P_(ENO2)-Ll_adhA^(RE1) 2 μ-ori, pUC ori, bla, G418r pGV2677 N.P_(PDC1-286)-Nc_ilvD2_coSc, crassa ilvD2P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6), P_(ENO2)-Ll_adhA^(RE1) 2 μ-ori,pUC ori, bla, G418r pGV2678 Acidobacteria P_(PDC1-286)-Ab_ilvD_coSc,bacterium P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6), P_(ENO2)-Ll_adhA^(RE1) 2μ-ori, pUC ori, bla, G418r pGV2679 AcaryochlorisP_(PDC1-286)-Am_ilvD_coSc, marina P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6),P_(ENO2)-Ll_adhA^(RE1) 2 μ-ori, pUC ori, bla, G418r pGV2680 Lyngbya spp.P_(PDC1-286)-Lsp_ilvD_coSc, P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6),P_(ENO2)-Ll_adhA^(RE1) 2 μ-ori, pUC ori, bla, G418r pGV2681 E. coliP_(PDC1-286)-Ec_ilvD_coKl, P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1-his6),P_(ENO2)-Ll_adhA^(RE1) 2 μ-ori, pUC ori, bla, G418r

TABLE 32 DHAD sequences disclosed in Example 18. SEQ ID NO SEQ ID NODHAD Abbreviation (DNA) (protein) L. lactis Ll_ilvD_coSc 155 18 S.cerevisiae ilv3ΔN20 Sc_ilv3_ΔN20 89 26 G. forsetii Gf_ilvD_coSc 90 17 S.erythraea Se_ilvD_coSc 91 19 F. tularensis Ft_ilvD_coSc 156 14 S.cerevisiae ilv3ΔN19 Sc_ilv3_ΔN19 157 163 S. cerevisiae ilv3ΔN23Sc_ilv3_ΔN23 158 164 N. crassa ilvD2 Nc_ilvD2_coSc 159 165 A. bacteriumAb_ilvD_coSc 92 16 A. marina Am_ilvD_coSc 160 166 Lyngbya spp.Lsp_ilvD_coSc 161 167 E. coli Ec_ilvD_coKl 162 168

Cloning techniques included digestion with restriction enzymes, gelpurification of DNA fragments (using the Zymoclean Gel DNA Recovery Kit,Cat# D4002, Zymo Research Corp, Orange, Calif.), ligation of two DNAfragments using the DNA Ligation Kit (Mighty Mix Cat# TAK 6023, ClontechLaboratories, Madison, Wis.), and bacterial transformations intocompetent E. coli cells (Xtreme Efficiency DH5α Competent Cells, Cat#ABP-CE-CCO2096P, Allele Biotechnology, San Diego, Calif.). Plasmid DNAwas purified from E. coli cells using the Qiagen QIAprep Spin MiniprepKit (Cat#27106, Qiagen, Valencia, Calif.).

Yeast media used for this example include YP medium (1% (w/v) yeastextract, 2% (w/v) peptone), YPD medium (YP medium containing 2% (w/v)glucose), YPD supplemented with glycerol and ethanol (YPD mediumcontaining 1% (v/v) 80% glycerol and 1% (v/v) ethanol. The antibioticG418 was added to agar plates to a final concentration of 0.2 g/L.Precultures were grown in YP medium supplemented with 5% glucose, 1%ethanol, and 0.2 g/L G418. Fermentations were carried out in YP mediumcontaining 8% glucose, 1% v/v of ergosterol and Tween-80 in 100%ethanol, 200 mM MES (pH 6.5), and 0.2 μg/mL G418.

A large patch of S. cerevisiae strain GEVO3868 was grown on an YPDplate. Cells from the patch were scraped from the plate, resuspended in2 mL YPD containing 1% v/v ethanol containing 1% v/v 80% glycerol andplaced in the 30° C. orbital shaker overnight. The following morning, 1mL of the overnight culture was used to inoculate 50 mL YPD containing1% ethanol containing 1% v/v 80% glycerol and returned to the 30° C.orbital shaker. After 6 hours, the cells were at an OD₆₀₀ of 0.55. Theywere diluted to an OD₆₀₀ of 0.1 in the same media and grown overnight at30° C. In the morning the cells were diluted to an OD₆₀₀ of 0.6, grownfor 3 hours at 30° C. until the OD₆₀₀ was 1.1, and the cells werecollected by centrifugation at 2700 rcf for 2 min at room temperature.The medium was removed, 50 mL sterile milliQ water was used to wash thecells, and the cells were centrifuged for 2 min at 2700 rcf at roomtemperature. After removing the supernatant, the cells were washed with25 mL sterile milliQ water and centrifuged at 2700 rcf for 2 min at roomtemperature. The supernatant was removed and the cells were resuspendedin 1 mL 100 mM lithium acetate. The cells were centrifuged for 10 sec,the supernatant removed, and the cells resuspended in 400 μL 100 mMlithium acetate. The cells were transformed as follows. First, a mixtureof plasmid DNA (final volume of 15 μl with sterile water), 72 μl 50%PEG, 10 μl 1M lithium acetate, and 3 μl of denatured salmon sperm DNA(10 mg/mL) was prepared for each transformation. In a sterile 1.5 mLtube, 15 μl of the cell suspension was added to the DNA mixture (100μl), and the transformation suspension was vortexed for 5 short pulses.The transformation was incubated for 30 min at 30° C., followed byincubation for 22 min at 42° C. The cells were collected bycentrifugation (18,000×g, 10 seconds, 25° C.). After removing thesupernatant, the cells were resuspended in 400 μl YPD. After anovernight recovery shaking at 30° C. and 250 RPM, the cells were spreadover selective plates, YPD containing 0.2 g/L G418. Transformants werethen single colony purified onto selective plates.

For fermentations, 3 mL cultures of GEVO3868 transformed with each 2pplasmid were started in YPD containing 1% ethanol containing 0.2 g/LG418 and incubated overnight at 30° C. and 250 RPM. There were threebiological replicates of each strain for 39 cultures total. After theOD₆₀₀ of these cultures were taken the next day, the appropriate amountof culture was used to inoculate 50 mL of YP with 5% glucose containing1% ethanol containing 0.2 g/L G418 (baffled flask) to an OD₆₀₀ ofapproximately 0.1. These cultures were incubated at 30° C. and 250 RPMovernight. The next day, the cultures containing the S. cerevisiaeilv3ΔN20, the S. cerevisiae ilv3ΔN19, and the S. cerevisiae ilv3ΔN23 didnot reach an OD₆₀₀ of 5 (0.6-2.4) so incubation continued for another 24h at 30° C. and 250 RPM. The remaining 30 cultures had reached an OD₆₀₀of approximately 5 and were centrifuged in 50 mL Falcon tubes at 2700rcf for 5 min at 25° C. The cells from the 30 cultures were resuspendedin 50 mL YP with 8% glucose, 1% (v/v) ethanol, ergosterol, Tween-80, 200mM MES (pH 6.5), and 0.2 g/L G418. The cultures were transferred to 250mL unbaffled flasks with closed screw caps and incubated at 30° C. and75 RPM. The next day, the remaining 9 cultures were at a higher OD₆₀₀(3-5) and prepared for the fermentation as described above. At 24 and 48h after transfer to 250 mL unbaffled flasks with closed screw caps,samples of each of the 39 flasks were taken to determine OD₆₀₀ andprepared for gas chromatography as follows. 2 mL of sample (per flask)was removed and OD₆₀₀ was determined. The remaining sample wascentrifuged for 10 min at maximum speed. 1 mL of the supernatant wasanalyzed by gas chromatography as described. For the final 72 htimepoint, the same procedures were used for measuring OD₆₀₀ andanalysis by gas chromatography. In addition samples were analyzed byhigh performance liquid chromatography. Cells were also prepared forenzyme assays. After 3×15 mL Falcon tubes per flask were weighed (totalof 117), 14 mL of the appropriate sample was transferred into the Falcontubes. After centrifugation at 3000×g for 5 min at 4° C., thesupernatant was removed and the cells washed in 3 mL cold, sterilewater. The tubes were centrifuged as per above for 2 min, thesupernatant removed, and the tubes reweighed to determine total cellweight. The Falcon tubes were stored at −80° C.

Analysis of organic acid metabolites was performed on an HP-1200 HPLCsystem equipped with two Restek RFQ 150×7.8 mm columns in series.Organic acid metabolites were detected using an HP-1100 UV detector (210nm) and refractive index. The column temperature was 60° C. This methodwas isocratic with 0.0180 N H₂SO₄ (in Milli-Q water) as mobile phase.Flow was set to 1.1 mL/min. Injection volume was 20 μL and run time was16 min. Analysis was performed using authentic standards (>99%, obtainedfrom Sigma-Aldrich, with the exception of 2,3-dihydroxyisovalerate(DHIV), which was custom synthesized according to Cioffi et al., 1980,Anal Biochem 104: 485 and a 5-point calibration curve.

Analysis of volatile organic compounds, including ethanol and isobutanolwas performed on a HP 5890, 6890 or 7890 gas chromatograph fitted withan HP 7673 Autosampler, a DB-FFAP column (J&W; 30 m length, 0.32 mm ID,0.25 μM film thickness) or equivalent connected to a flame ionizationdetector (FID). The temperature program was as follows: 230° C. for theinjector, 300° C. for the detector, 100° C. oven for 1 minute, 70°C./minute gradient to 230° C., and then hold for 2.5 min. Analysis wasperformed using authentic standards (>99%, obtained from Sigma-Aldrich,and a 5-point calibration curve with 1-pentanol as the internalstandard.

For DHAD activity assays cells were thawed on ice and resuspended inlysis buffer (50 mM Tris pH 8.0 and 5 mM MgSO₄) for a 20% cellsuspension by mass. 1000 μl of glass beads (0.5 mm diameter) were addedto a 1.5 ml Eppendorf tube and 875 μl of cell suspension was added.Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc.Newtown, Pa.), mixing 6×1 min each at full speed with 1 min incubationson ice between each bead-beating step. The tubes were centrifuged for 10min at 23,500×g at 4° C. and the supernatant was removed for use. Theselysates were held on ice until assayed. Yeast lysate proteinconcentration was determined using the BioRad Bradford Protein AssayReagent Kit (Cat#500-0006, BioRad Laboratories, Hercules, Calif.) andusing BSA for the standard curve. Briefly 10 μL standard or lysate wereadded into a microcentrifuge tube. The samples were diluted to fit inthe linear range of the standard curve (1:40). 500 μL of diluted andfiltered Bio-Rad protein assay dye was added to the blank and samplesand then vortexed. Samples were incubated at room temperature for 6 min,transferred into cuvettes and the OD₅₉₅ was determined in aspectrophotometer. The linear regression of the standards was then usedto calculate the protein concentration in each sample. For DHAD assaystechnical triplicates were performed for each sample. In addition, a nolysate control with lysis buffer was performed. To assay each sample, 10μL of an appropriate dilution of lysate in assay buffer was mixed with90 μL of assay buffer (5 μL of 0.1 M MgSO₄, 10 μL of 0.1 M DHIV, and 75μL 50 mM Tris pH 8.0), and incubated in a thermocycler for 30 minutes at35° C., then at 95° C. for 5 minutes. Cell debris and precipitant wereremoved from the samples by centrifugation at 3000×g for 5 min.

Finally, 75 μL of supernatant was transferred to new PCR tubes andanalyzed by Liquid Chromatography for the 2-keto-isovalerate (KIV)product. DNPH reagent (12 mM 2,4-Dinitrophenyl Hydrazine 20 mM CitricAcid pH 3.0 80% Acetonitrile 20% MilliQ H₂O) was added to each sample ina 1:1 ratio. Samples were incubated for 30 min at 70° C. in athermo-cycler (Eppendorf, Mastercycler). Analysis of KIV was performedon an HP-1200 High Performance Liquid Chromatography system equippedwith an Eclipse XDB C-18 reverse phase column (Agilent) and a C-18reverse phase column guard (Phenomenex). Ketoisovalerate was detectedusing an HP-1100 UV detector (360 nm). The column temperature was 50° C.This method was isocratic with 70% acetonitrile 2.5% phosphoric acid(4%), 27.5% water as mobile phase. Flow was set to 3 mL/min. Injectionsize was 10 μL and run time was 2 min.

The data at 72 hours are summarized in Table 33. The data demonstratesthat the DHADs contained in plasmids pGV2635, 2677, 2674, 2672, 2673 and2676 led to production of isobutanol titers of at least 2.5 g/L and areconsidered to be significantly active in the cytosolic isobutanolpathway. The DHADs contained in plasmids pGV2675, 2681, 2680, 2678,2679, 2671, and 2676 led to production of isobutanol titers below 2.5g/L and are considered to be inactive or poorly active in the cytosolicisobutanol pathway.

TABLE 33 Isobutanol production with selected DHADs. Plasmid Isobutanolproduced DHAD activity (DHAD Gene) OD₆₀₀ [g/L] (U/mg) pGV2635 8.6 ± 0.69.02 ± 0.28 0.62 ± 0.01 (L. lactis) pGV2677 9.4 ± 0.6 6.30 ± 0.85 0.42 ±0.02 (N. crassa) pGV2674 7.5 ± 0.7 6.22 ± 0.31 0.30 ± 0.00 (F.tularensis) pGV2672 8.1 ± 0.6 6.10 ± 0.26 0.20 ± 0.00 (G. forsetii)pGV2673 8.0 ± 1.1 3.23 ± 0.12 0.03 ± 0.00 (S. erythraea) pGV2676 5.2 ±0.2 2.67 ± 0.06 0.02 ± 0.00 (S. cerevisiae ilv3ΔN23) pGV2675 5.0 ± 0.22.27 ± 0.16 0.09 ± 0.00 (S. cerevisiae ilv3ΔN19) pGV2681 6.9 ± 0.6 2.21± 0.09 0.03 ± 0.00 (E. coli) pGV2680 6.9 ± 1.3 2.13 ± 0.09 0.02 ± 0.00(Lyngbya spp.) pGV2678 7.5 ± 0.2 2.06 ± 0.17 0.03 ± 0.00 (Acidobacteria)pGV2679 7.5 ± 0.6 2.05 ± 0.06 0.03 ± 0.00 (A. marina) pGV2671 5.5 ± 0.01.92 ± 0.03 0.44 ± 0.01 (S. cerevisiae) pGV2663 6.7 ± 0.2 1.53 ± 0.180.01 ± 0.01 (none)

Example 19 Overexpression of the L. lactis ilvD in K. lactis and K.Marxianus

The purpose of this example is to demonstrate activity of L. lactis DHADin K. lactis and in K. marxianus.

Strains, plasmids, and sequences disclosed herein are listed in Tables34, 35, and 36, respectively.

TABLE 34 Genotype of strains disclosed in Example 19. GEVO NumberGenotype K. marxianus strain K. marxianus NRRL-Y-7571 ura3-delta2GEVO2504 pdc1Δ::Ll.kivd2 coSc. P_(TDH3): Dm_ADH:P_(FBA1):URA3:P_(Sc)_FBA1:31COX4_MTS:Bs_alsS1_coSc K. marxianus strain ura3-delta2pdc1Δ::Δ::{Ll_kivd2 GEVO2543 co:P_(Sc)_TDH3:Ec_ilvC^(Q11V) coSC:P_(Sc)_TPI1:G418^(R):P_(Sc)_CUP1:Bs_alsS1_coSc} K. marxianus strainura3-delta2 pdc1Δ::{Ll_kivd2 GEVO2598 co:P_(Sc)_TDH3:Ec_ilvC^(Q110V)coSC: P_(Sc)_TPI1:G418^(R):P_(Sc)_CUP1:Bs_alsS1_coSc} + randomintegration of {P_(Sc)_TEF1:Ll_ilvD_coSc URA3} K. lactis strain MATalphauraA1 trp1 leu2 lysA1 ade1 GEVO1287 lac4-8 [pKD1] ATCC 200826

TABLE 35 Plasmids disclosed in Example 19. Plasmid Name RelevantGenes/Usage Genotype pGV2271 Empty 1.6 micron 1.6 μ ori, bla, hygroRvector that can be maintained in K. lactis. Encodes hygromycinresistance. pGV2273 1.6 micron vector for P_(TDH3): Ec_ilvC_P2D1-A1,expression of KARI, P_(TEF1): Ll_ilvD_coSc, P_(PGK1): KIVD, DHAD and ADHLl_kivD2_coEc, in K. lactis P_(ENO2): Ll_adhA 1.6 μ ori, bla, HygroRpGV2069 2 micron plasmid for P_(TDH3): Ec_ilvC_coScQ^(110V), expressionof KIVD, P_(TEF1): Ll_ilvD_coSc, P_(PGK1): DHAD, KARI, and ALSLl_kivD2_coEc, P_(CUP1): in K. marxianus Bs_alsS1_coSc, P_(ENO2):Dm_adhA, 2 μ ori, bla, G418 pGV1855 2 micron plasmid for expressionP_(TEF1): Ll_ilvD, 2 μ ori, of DHAD in K. marxianus bla, URA

TABLE 36 Amino acid and nucleotide sequences of enzymes and genesdisclosed in Example 19. Corresponding Protein Enz. Source Gene (SEQ IDNO) (SEQ ID NO) ALS B. subtilis Bs_alsS1_coSc Bs_AlsS1_coSc (SEQ ID NO:144) (SEQ ID NO: 169) KARI E. coli Ec_ilvC_coSc^(Q110V)Ec_IlvC_coSc^(Q110V) (SEQ ID NO: 98) (SEQ ID NO: 170) E.coliEc_ilvC_coSc^(P2D1-A1) Ec_ilvC_coSc^(P2D1-A1) (SEQ ID NO: 171) (SEQ IDNO: 172) KIVD L. lactis Ll_kivd2_coEc Ll_Kivd2_coEc (SEQ ID NO: 99) (SEQID NO: 173) DHAD L. lactis Ll_ilvD_coSc Ll_IlvD_coSc (SEQ ID NO: 155)(SEQ ID NO: 18) ADH L. lactis Ll_adhA Ll_adhA (SEQ ID NO: 174) (SEQ IDNO: 175) D. melanogaster Dm_adh Dm_adh (SEQ ID NO: 116) (SEQ ID NO: 176)

To generate GEVO2543, GEVO2504 was transformed with pGV2069 to integrateinto the genome three genes: Bs_alsS1_coSc (SEQ ID NO: 144),Ec_ilvC_coSc^(Q110V) (SEQ ID NO: 98), and LI_kivd2_coEc (SEQ ID NO: 99).To generate GEVO2598, GEVO2543 was transformed pGV1855 to integrate theL. lactis ilvD gene which was codon optimized for S. cerevisiae (genesequence SEQ ID NO: 155, also referred to as LI_ilvD_coSc; proteinsequence SEQ ID NO: 18) into the chromosome. GEVO1287 was transformedwith either pGV2271 (control plasmid) or pGV2273, which containsLI_ilvD_coSc.

GEVO2543, GEVO2598 and GEVO1287 transformed with pGV2271 or pGV2273 wereinoculated into 3 mL of YPD (for GEVO2543 and GEVO2598) or YPDsupplemented with 0.1 g/L hygromycin (for GEVO1287) for an overnightculture. After approximately 18 hours, a 50 ml YPD culture in a baffled250 ml shake flask was inoculated to 0.15 OD₆₀₀ and shaken at 250 rpmsfor approximately 9 hours. Next, DHAD activity and proteinconcentrations were measured.

Over-expression of the L. lactis ilvD gene resulted in an increase inDHAD activity (U/mg total cell lysate protein). Table 37 shows the DHADactivity (U/mg total cell lysate protein) averages from technicaltriplicates comparing strains expressing the L. lactis DHAD to strainsnot expressing the L. lactis DHAD gene.

TABLE 37 DHAD activity in whole cell yeast lysates. Strain Activity[mU/mg] K. marxianus strain GEVO2543 (no DHAD) 0.010 ± 0.002 K.marxianus strain GEVO2598 (DHAD) 0.016 ± 0.001 K. lactis strainGEVO1287 + pGV2271 (No DHAD) 0.052 ± 0.003 K. lactis strain GEVO1287 +pGV2273 (DHAD) 0.122 ± 0.011

Example 20 L. lactis ilvD Activity is Localized to the Yeast Cytosol

The purpose of this example is to demonstrate that the Lactococcuslactis ilvD protein localizes to the cytosol when expressed in a yeaststrain.

The S. cerevisiae strain GEVO1187 (S. cerevisiae CEN.PK2, MATa ura3 leu2his3 trp1 ADE2) was transformed with plasmid pGV2484, a 2 micron plasmidexpressing the L. lactis ilvD gene which was codon optimized for S.cerevisiae (gene sequence SEQ ID NO: 155, also referred to asLI_ilvD_coSc; protein sequence SEQ ID NO: 18) under the S. cerevisiaeTEF1 promoter (P_(TEF1):LI_ilvD_coSc, 2μ ori, bla, G418R). Briefly, thestrain was grown in YPD to an OD₆₀₀ of 0.6-0.8. Cells were washed inH₂0, and then resuspended in 100 mM Lithium acetate. In a 1.5 mL tube,15 μL of the cell suspension was added to a mixture of DNA (final volumeof 15 μl with sterile water), 72 μl 50% PEG, 10 μl 1M lithium acetate,and 3 μl of denatured salmon sperm DNA (10 mg/mL). The transformationsuspension was vortexed for 5 short pulses. The mixture was incubated at30° C. for 30 minutes, followed by incubation for 22 minutes at 42° C.The cells were collected by centrifugation (18,000×g, 10 seconds, 25°C.). The cells were resuspended in 1 ml YPD medium (1% (w/v) yeastextract, 2% (w/v) peptone, 2% (w/v) glucose, pH 5) and after anovernight recovery shaking at 30° C. and 250 rpms, the cells were spreadover YPD agar plates supplemented with 0.2 g/L G418. Transformants werethen single colony purified onto G418 selective plates.

All isolations of crude mitochondrial fractions were performed induplicate. GEVO1187 and GEVO1187 transformed with pGV2484 were eachgrown in 100 mL of YPG medium (1% (w/v) yeast extract, 2% (w/v) peptone,3% (v/v) glycerol, pH5) overnight at 30° C. and 250 rpm. This overnightculture was used to inoculate 840 mL of YPG in a 2800 mL baffled flaskat an OD₆₀₀ of 0.03, and cells were grown at 30° C. and 250 rpm for20-28 h. At an OD₆₀₀ of about 2.0, cells were harvested bycentrifugation at 3000×g for 5 minutes, resuspended in 100 mL H₂Ofollowed by centrifugation at 3000×g for 5 minutes. Cells were incubatedin 2 mL/g CWW (cell wet weight) of DTT buffer (100 mM Tris-H₂SO₄ pH 9.4,10 mM DTT) for 20 minutes at 30° C. Cells were resuspended in 7 mL/g CWWZymolyase buffer (1.2 M sorbitol, 20 mM Potassium phosphate pH 7.4) andthen centrifuged at 3000×g for 5 minutes. Cells were spheroplasted byincubating in Zymolyase buffer with Zymolyase (Seikagaku BiobusinessCorporation #120491-1; 3 mg/g CWW) for 45 minutes at 30° C. on a rockingplatform. 100 OD of spheroplasts were set aside for whole cell lysatepreparation (see below). Spheroplasts were resuspended in Zymolyasebuffer and centrifuged at 3000×g for 5 minutes before resuspension in6.5 mL/g CWW homogenization buffer (chilled to 4° C.; 6.5 mL/g 0.6 Msorbitol, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM PMSF, 0.2% (w/v) BSA).Spheroplasts were homogenized on ice with 15 strokes of a pre-chilledglass-Teflon homogenizer (40 mL capacity), and the sample was diluted2-fold with homogenization buffer. Cell debris and nuclei were pelletedby serial supernatant centrifugations of 1500×g for 5 minutes, and4000×g for 5 minutes. The mitochondrial fraction was isolated bycentrifugation at 12,000×g for 15 minutes. The crude mitochondrialpellet was resuspended in 10 mL SEM buffer (250 mM sucrose, 1 mM EDTA,10 mM MOPS-KOH pH 7.2), centrifuged at 4000×g for 5 minutes to furtherremove cellular debris and nuclei before recovering the mitochondrialfraction by centrifugation at 12,000×g for 15 minutes. The mitochondrialfraction may contain markers of the plasma membrane, the endoplasmicreticulum, and vacuoles in addition to markers of the mitochondria.Mitochondrial pellet was resuspended in 750 μL SEM Buffer+ProteaseArrest (GBiosciences #786-108).

Preparation of whole cell yeast lysates was performed using the 100 ODsof yeast cells set aside after spheroplasting (see above) byresuspending cells in 20% (w/v) SEM Buffer+1× Protease Arrest(GBiosciences #786-108). 1000 μl of glass beads (0.5 mm diameter) wereadded to a 1.5 ml eppendorf tube, and 875 μl of cell suspension wasadded. Yeast cells were lysed using a Retsch MM301 mixer mill (RetschInc. Newtown, Pa.), mixing 6×1 min each at full speed with 1 minincubations on ice between each bead-beating step. The tubes werecentrifuged for 10 min at 23,500×g at 4° C., the supernatant wasremoved, aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.

The resuspended mitochondrial fraction (see above) was added to 1000 μlof glass beads (0.1 mm diameter) in a 1.5 ml Eppendorf tube. Additionalbuffer was added if necessary to fill the tube completely. Themitochondrial fraction was lysed using a Retsch MM301 mixer mill (RetschInc. Newtown, Pa.), mixing 3×1 minute each at full speed with 1 minuteincubations on ice between each bead-beating step. The tubes werecentrifuged for 10 min at 23,500×g at 4° C., the supernatant wasremoved, aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.

Whole cell yeast lysate and mitochondrial fraction lysate proteinconcentration was determined using the BioRad Bradford Protein AssayReagent Kit (Cat#500-0006, BioRad Laboratories, Hercules, Calif.) andusing BSA for the standard curve. Briefly, 10 μL standard or lysate wereadded into a microcentrifuge tube. The samples were diluted to fit inthe linear range of the standard curve (1:10-1:40). 500 μL of dilutedand filtered Bio-Rad protein assay dye was added to the blank andsamples and then vortexed. Samples were incubated at room temperaturefor 6 mins, transferred into cuvettes and the OD₅₉₅ was determined in aspectrophotometer. The linear regression of the standards was then usedto calculate the protein concentration in each sample.

Three samples of each of the mitochondrial and whole cell yeast lysateswere assayed for DHAD activity, along with no lysate controls. Table 38shows the DHAD activity (U/mg protein) averages from duplicate culturescomparing strains GEVO1187 (no DHAD expression) to GEVO1187 transformedwith pGV2484 (L. lactis DHAD expressed from pGV2484). DHAD activity wasmeasured in the whole cell yeast lysate and the mitochondrial fractionlysate. Expression of DHAD from pGV2484 resulted in about a 7-foldincrease in DHAD activity in the whole cell yeast lysate. Expression ofDHAD from pGV2484 did not affect DHAD activity localized to themitochondrial fraction. Subtracting the background activity in theGEVO1187 whole cell yeast lysate of 0.27 mU/mg from the activity in thewhole cell yeast lysate of GEVO1187 transformed with pGV2484 of 1.87mU/mg shows an increase in 1.60 mU/mg. These data suggest that L. lactisDHAD activity does not localize to the organellar structures harvestedin the mitochondrial fraction, and is therefore cytosolic when expressedin a yeast strain.

TABLE 38 DHAD activity in whole cell yeast lysates and mitochondrialfraction lysates. Activity Strain Lysate [mU/mg] GEVO1187 Whole cell0.27 ± 0.07 GEVO1187 transformed with pGV2484 Whole cell 1.87 ± 0.14GEVO1187 Mitochondrial 3.76 ± 0.01 GEVO1187 transformed with pGV2484Mitochondrial 3.85 ± 0.13

Example 21 Overexpression of the L. lactis ilvD in Issatchenkiaorientalis

The purpose of this example is to demonstrate cytosolic activity of L.lactis DHAD in I. orientalis.

An engineered strain derived from the wild-type I. orientalis strainATCC PTA-6658 was further modified to contain copies of all fiveisobutanol pathway genes integrated into the chromosome. First, bothalleles of the PDC1 locus were deleted in series (See e.g.WO/2007/106524, which is herein incorporated by reference in itsentirety). The deletion event also simultaneously integrated a copy ofB. subtilis alsS gene and a copy of the L. lactis kivD gene which encodeSEQ ID NOs: 169 and 173, respectively. This resulted in a Pdc-strainwith two integrated copies of the B. subtilis alsS gene and twointegrated copies of the L. lactis kivD gene (pdc1Δ:LI_kivD: Bs_alsSpdc1Δ:LI_kivD: Bs_alsS). This strain was further engineered to delete asingle allele of the GPD1 locus (See e.g. WO/2007/106524). The deletionevent also simultaneously integrated a single copy of the L. lactisadhA^(RE1), the E. coli ilvC^(P2D1-A1), and L. lactis ilvD which encodethe proteins shown in SEQ ID NOs: 177, 172, and 18, respectively. Thisresults in a Pdc− Gpd+ strain with one integrated copy of theLI_adhA^(RE1), Ec_ilvC^(P2D1-A1), and LI_ilvD genes(GPD1/gpd1Δ:[LI_adhA^(RE1): Ec_ilvC^(P2D1-A1): URA3:LI_ilvD]). Thisstrain is GEVO4306 (Table 39).

To generate a control strain which does not express the pathway genes,both alleles of the PDC1 locus were deleted in series but with nosimultaneous integration of heterologous genes. Next one of the two GPD1alleles was deleted with no simultaneous integration of heterologousgenes. The resulting control strain is GEVO4308 (pdc1Δ::loxP/pdc1Δ::loxPGPD1/gpd1Δ::loxP:URA3:loxP) (Table 39).

TABLE 39 Genotype of strains disclosed in Example 21. GEVO NumberGenotype 4306 pdc1Δ::[Ll_kivD: Bs_alsSl pdc1Δ::Ll_kivD: Bs_alsS]GPD1/gpd1Δ::[Ll_adhA^(RE1): Ec_ilvC^(P2D1-A1): URA3Ll_ilvD] 4308pdc1Δ::loxP/pdc1Δ::loxP GPD1/gpd1Δ::loxP:URA3:loxP

Over-expression of the L. lactis ilvD gene resulted in an increase inDHAD activity (U/mg total cell lysate protein). Table 40 shows the DHADactivity (U/mg total cell lysate protein) averages from technicaltriplicates comparing the strain expressing the L. lactis DHAD gene tothe strain not expressing the L. lactis DHAD gene. Expression of the L.lactis ilvD gene, when expressed with the remainder of the isobutanolpathway, resulted in isobutanol production as seen in Table 40.

TABLE 40 DHAD activity in whole cell yeast lysates and isobutanol titerafter 72 hr fermentation. Strain Activity [mU/mg] Isobutanol titer g/LGEVO4306 0.041 ± 0.009 0.56 ± 0.01 GEVO4308 0.012 ± 0.002 0.00 ± 0.00

Example 22 Cytosolic ALS Homologs that Support Isobutanol Production

This example demonstrates isobutanol production using expression ofcytosolically localized ALS genes in the presence of the rest of theisobutanol pathway. The ALS genes were integrated into the PDC1 locus ofS. cerevisiae strain GEVO1187 and isobutanol production was achieved byexpression from plasmid of the other genes in the isobutanol pathway.Isobutanol production in strains carrying the ALS genes from T.atroviride (Ta_ALS) and T. stipitatus (Ts_ALS) was compared toisobutanol production in strains carrying the ALS gene from B. subtilis.Plasmids described in this example are listed in Table 41.

TABLE 41 Plasmids disclosed in Example 22. Plasmid name RelevantGenes/Usage Genotype pGV1730 Integration plasmid that will integrate SeeTable 14. P_(CUP1-1):Bs_alsS2 into PDC1 using digestion was the withNruI for targeting. This parent vector for cloning the ALS homologs.pGV1773 Vector with Bacillus subtilis AlsS P_(PDC1):Bs_AlsS1_coSc, codonoptimized for S. cerevisiae. P_(TDH3):Ll_kivD, P_(ADH1):Sc_ADH7_coSc,URA3 5′-end, pUC ORI, kan^(R). pGV1802 DNA2.0 plasmid carrying theTa_ALS_coSc in DNA Trichoderma atrovirideALS. 2.0 vector pGV1803 DNA2.0plasmid carrying the Ts_ALS_coSc in DNA Talaromyces stipitatus ALS. 2.0vector pGV2082 High copy 2 μ plasmid with 4 Ec_ilvC_coSc^(Q110V),isobutanol pathway genes Ll_ilvD_coSc, without an ALS gene.Ll_kivD2_coEc, and Dm_ADH, 2 μ ori, bla, G418R. pGV2114 Integrationplasmid that will integrate See Table 14. into PDC1 using digestion withNruI for targeting. It carries the Bacillus subtilis AlsS gene codonoptimized for S. cerevisiae. pGV2117 Integration plasmid that will SeeTable 14. integrate into PDC1 using digestion with NruI for targeting.It carries the Trichoderma atroviride ALS gene codon optimized for S.cerevisiae. pGV2118 Integration plasmid that will See Table 14.integrate into PDC1 using digestion with NruI for targeting. It carriesthe Talaromyces stipitatus ALS gene codon optimized for S. cerevisiae.

Strains with integrated ALS genes expressed from the CUP1 promoter weretransformed with pGV2082 (which carries the other 4 isobutanol pathwaygenes Ec_ilvC_coScQ110V (SEQ ID NO: 98), LI_ilvD (SEQ ID NO: 155),LI_kivd2_coEc (SEQ ID NO: 99), and Dm ADH (SEQ ID NO: 116).

GEVO2618, GEVO2621, and GEVO2622 (see Table 13) were each transformedwith pGV2082. Control strains GEVO2280 (B. subtilis alsS2) (Table 13)and GEVO1187 (no ALS) (Table 13) were also transformed with pGV2082.

Fermentations of the transformed strains GEVO1187, GEVO2280, GEVO2618,GEVO2621, GEVO2622 were performed. Strains encoding the ALS from T.atroviride (SEQ ID NO: 71) and T. stipitatus (SEQ ID NO: 72) producedmore isobutanol than the strain containing the B. subtilis als2. Thestrain containing Bs_Als1_coSc produced the most isobutanol. Table 42shows the final OD, glucose consumption, and isobutanol titer for eachof the strains. The integration of the cytosolic genes Ta_ALS_coSc andTs_ALS_coSc led to production of isobutanol that was in each case 6-foldabove that of a strain without an integrated ALS gene, demonstratingthat these strains are producing isobutanol using a cytosolic pathway.

TABLE 42 Results of fermentations with cytosolic ALS homologs at 72 hrs.Strain OD₆₀₀ Glucose consumed g/L Isobutanol produced g/L GEVO1187 10.9± 0.3  233 ± 36 0.3 ± 0.0 GEVO2280 9.9 ± 0.3 274 ± 26  1.3 ± 0.11GEVO2618 9.4 ± 0.2 138 ± 9  2.6 ± .09 GEVO2621 9.9 ± 0.3 161 ± 52 1.9 ±.18 GEVO2622 10.8 ± 0.6  182 ± 47 1.8 ± .15

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodthere from as modifications will be obvious to those skilled in the art.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

What is claimed is:
 1. A recombinant yeast microorganism comprising anisobutanol producing metabolic pathway, wherein said isobutanolproducing metabolic pathway comprises the following substrate to productconversions: (i) pyruvate to acetolactate; (ii) acetolactate to2,3-dihydroxyisovalerate; (iii) 2,3-dihydroxyisovalerate toα-ketoisovalerate; (iv) α-ketoisovalerate to isobutyraldehyde; and (v)isobutyraldehyde to isobutanol; wherein a) the enzyme that catalyzes asubstrate to product conversion of pyruvate to acetolactate is anacetolactate synthase; b) the enzyme that catalyzes a substrate toproduct conversion of acetolactate to 2,3-dihydroxyisovalerate is aketol-acid reductoisomerase derived from Lactococcus lactis; c) theenzyme that catalyzes a substrate to product conversion of2,3-dihydroxyisovalerate to α-ketoisovalerate is a dihydroxy aciddehydratase; d) the enzyme that catalyzes a substrate to productconversion of α-ketoisovalerate to isobutyraldehyde is anα-ketoisovalerate decarboxylase; and e) the enzyme that catalyzes asubstrate to product conversion of isobutyraldehyde to isobutanol is analcohol dehydrogenase.
 2. The recombinant yeast microorganism of claim1, wherein said acetolactate synthase is derived from a bacterialorganism.
 3. The recombinant yeast microorganism of claim 2, whereinsaid bacterial organism is Bacillus subtilis.
 4. The recombinant yeastmicroorganism of claim 1, wherein said ketol-acid reductoisomerase is anNADH-dependent ketol-acid reductoisomerase.
 5. The recombinant yeastmicroorganism of claim 1, wherein said dihydroxy acid dehydratasecomprises the amino acid sequence P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27),wherein X is any natural or non-natural amino acid.
 6. The recombinantyeast microorganism of claim 5, wherein said dihydroxy acid dehydrataseenzyme is derived from a bacterial organism.
 7. The recombinant yeastmicroorganism of claim 6, wherein said bacterial organism is Lactococcuslactis.
 8. The recombinant yeast microorganism of claim 1, wherein saidα-ketoisovalerate decarboxylase is derived from a bacterial organism. 9.The recombinant yeast microorganism of claim 8, wherein said bacterialorganism is Lactococcus lactis.
 10. The recombinant yeast microorganismof claim 1, wherein said alcohol dehydrogenase is derived from abacterial organism.
 11. The recombinant yeast microorganism of claim 10,wherein said bacterial organism is Lactococcus lactis.
 12. Therecombinant yeast microorganism of claim 1, wherein the recombinantyeast microorganism has been engineered to inactivate one or moreendogenous pyruvate decarboxylase (PDC) genes.
 13. The recombinant yeastmicroorganism of claim 12, wherein said one or more endogenous PDC genesis selected from PDC1, PDC5, and PDC6.
 14. The recombinant yeastmicroorganism of claim 1, wherein the recombinant yeast microorganismhas been engineered to inactivate one or more endogenousglycerol-3-phosphate dehydrogenase (GPD) genes.
 15. The recombinantyeast microorganism of claim 14, wherein said one or more endogenous GPDgenes is selected from GPD1 and GPD2.
 16. The recombinant yeastmicroorganism of claim 1, wherein the recombinant yeast microorganism isa yeast microorganism of the Saccharomyces clade.
 17. The recombinantyeast microorganism of claim 16, wherein said yeast microorganism of theSaccharomyces clade is S. cerevisiae.
 18. A method of producingisobutanol, comprising: (a) providing a recombinant yeast microorganismaccording to claim 1; and (b) cultivating said recombinant yeastmicroorganism in a culture medium containing a feedstock providing thecarbon source, until a recoverable quantity of the isobutanol isproduced.